Modification of saponins

ABSTRACT

Methods for the enzymatic modification of saponins, products made thereby, uses of said products and also to other associated aspects. The saponins may be extracts of Quillaja species, such as extracts of Quillaja saponaria Molina.

TECHNICAL FIELD

The present application generally relates to saponins, in particular methods for the enzymatic modification of saponins, products made thereby, uses of said products and also to other associated aspects. The present application further relates to glucosidases and rhamnosidases, in particular mutated glucosidases and rhamnosidases which may be of use in methods for the enzymatic modification of saponins. The saponins may be obtainable from Quillaja species, including extracts obtainable from Quillaja species, such as extracts of Quillaja saponaria Molina.

BACKGROUND

Saponins are steroid or terpenoid glycosides. They have a broad range of uses from fire extinguisher foams to food additives and immunostimulants (Reichert, 2019).

Quillaja extract (E 999) is currently approved by the European Food Safety Authority under EU Regulation 1129/2011 as a food additive in flavoured drinks (14.1.4), cider and perry (14.2.3). Quillaja extract (E 999) is described as being obtained by aqueous extraction of the milled inner bark or wood of Quillaja saponaria, or other Quillaja species. It is also described as containing a number of triterpenoid saponins consisting of glycosides of quillaic acid. Sugars—including glucose, galactose, arabinose, xylose, and rhamnose—are also said to be present, along with tannin, calcium oxalate and other minor components. (EFSA Journal 2019 17(3):5622)

Saponins have been of interest as immunostimulants for many decades (see, for example, Hyslop, 1969). Quil A is a saponin fraction derived from an aqueous extract from the bark of Quillaja saponaria (Dalsgaard, 1974). Quil A itself contains a plurality of components with the four most predominant Quil A fractions purified by reverse phase chromatography, namely QS-7, QS-17, QS-18 and QS-21, all having immunostimulatory activity although varying in haemolytic activity and toxicity (Kensil, 1991; Kensil, 1995). The main saponin fraction, QS-18, was found to be highly toxic in mice but saponin fractions QS-7 and QS-21 were far less toxic. QS-21, being more abundant than QS-7, has been the most widely studied saponin adjuvant (Ragupathi, 2011).

Liquid chromatography/mass spectrometry analysis of Quillaja saponaria bark water/methanol extracts has revealed over 100 saponins, many of which have been assigned structures (Nyberg, 2000; Nyberg, 2003; Kite, 2004).

Quillaja brasiliensis (A St.-Hil & Tul) Mart. extracts have been described, with the identity of various components therein determined by mass spectrometry. Many saponin components in Quillaja brasiliensis extracts correspond to saponins found in Quillaja saponaria extracts (Wallace, 2017; Wallace, 2019) and Quillaja brasiliensis extracts have also been shown to have immunostimulant effects (Cibulski, 2018; Yendo, 2017).

The Adjuvant System 01 (AS01) is a liposome-based adjuvant which contains two immunostimulants, 3-O-desacyl-4′-monophosphoryl lipid A (3D-MPL) and QS-21 (Garcon, 2011; Didierlaurent, 2017). 3D-MPL is a non-toxic derivative of the lipopolysaccharide from Salmonella minnesota. AS01 is included in vaccines for malaria (RTS, S-Mosquirix™) and Herpes zoster (HZ/su—Shingrix™), and in multiple candidate vaccines. AS01 injection results in rapid and transient activation of innate immunity in animal models. Neutrophils and monocytes are rapidly recruited to the draining lymph node (dLN) upon immunization. Moreover, AS01 induces recruitment and activation of MHCII^(high) dendritic cells (DC), which are necessary for T cell activation (Didierlaurent, 2014). Some data are also available on the mechanism of action of the components of AS01. 3D-MPL signals via TLR4, stimulating NF-κB transcriptional activity and cytokine production and directly activates antigen-presenting cells (APCs) both in humans and in mice (De Becker, 2000; Ismaili, 2002; Martin, 2003; Mata-Haro, 2007). QS-21 promotes high antigen-specific antibody responses and CD8⁺ T-cell responses in mice (Kensil, 1998; Newman, 1992; Soltysik, 1995) and antigen-specific antibody responses in humans (Livingston, 1994). Because of its physical properties, it is thought that QS-21 might act as a danger signal in vivo (Lambrecht, 2009; Li, 2008). Although QS-21 has been shown to activate ASC-NLRP3 inflammasome and subsequent IL-1β/IL-18 release (Marty-Roix, 2016), the exact molecular pathways involved in the adjuvant effect of saponins have yet to be clearly defined.

Extracts of Quillaja saponaria are commercially available, including fractions thereof with differing degrees of purity such as Quil A, Fraction A, Fraction B, Fraction C, QS-7, QS-17, QS-18 and QS-21.

The enzymatic hydrolysis of Quil-A by Rapidase® Revelation Aroma has been described during the development of a quality control method to provide a degraded reference sample (Lecas, 2021).

Availability of saponins is constrained, particularly those obtained from rarer plants or where saponins of interest are present in relatively low amounts. Furthermore, separation of certain saponins from other components, particularly other saponin components which may have similar structures, can be burdensome. Consequently, there remains a need for new methods which may improve the yield of saponins of interest and/or facilitate removal of undesired saponin components.

Modestobacter marinus glucosidase (Uniparc reference UPI000260A2FA, Uniprot reference I4EYD5) is a naturally occurring glucosidase. There remains a need for further glucosidases which may have improved properties.

Kribbella flavida rhamnosidase (Uniparc reference UPI00019BDB13, Uniprot reference D2PMT) is a naturally occurring rhamnosidase. There remains a need for further rhamnosidases which may have improved properties.

SUMMARY OF THE INVENTION

The present invention provides a method for making a product saponin, said method comprising the step of enzymatically converting a starting saponin to the product saponin. Suitably the method uses a polypeptide of the invention, such as an engineered glucosidase polypeptide or an engineered rhamnosidase polypeptide.

Also provided is a method for increasing the amount of a product saponin in a composition, said method comprising the step of enzymatically converting a starting saponin to the product saponin. Suitably the method uses a polypeptide of the invention, such as an engineered glucosidase polypeptide or an engineered rhamnosidase polypeptide.

Further provided is a method for reducing the amount of a starting saponin in a composition, said method comprising the step of enzymatically converting the starting saponin to a product saponin. Suitably the method uses a polypeptide of the invention, such as an engineered glucosidase polypeptide or an engineered rhamnosidase polypeptide.

The use of a glycosidase for enzymatically converting a starting saponin to a product saponin is also provided by the invention. Suitably the glycosidase is a polypeptide of the invention, such as an engineered glucosidase polypeptide or an engineered rhamnosidase polypeptide.

Additionally provided is a method for identifying a candidate enzyme having beta exo glucosidase activity, comprising selecting an enzyme comprising, such as consisting of: (i) an amino acid sequence according to SEQ ID No. 262, 208, 63, 229, 250, 5, 101, 207, 169, 247, 302, 324, 319, 9, 240, 325, 338, 850, 879, 868, 826, 804, 888, 881, 891, 816, 827, 857, 853, 842, 814, 886, 885, 838, 829, 808, 828, 870, 873, 844, 882, 874, 825, 824, 823, 810, 894, 849, 803, 890, 841, 832, 830, 845, 871, 837, 883 or 809 or functional variants thereof; or (ii) an amino acid sequence according to SEQ ID No. 262, 208, 63, 229, 250, 5, 101, 207, 169, 247, 302, 324, 319, 9, 240, 325, 338, 850, 879, 868, 826, 804, 888, 881, 891, 816, 827, 857, 853, 842, 814, 886, 885, 838, 829, 808, 828, 870, 873, 844, 882, 874, 825, 824, 823, 810, 894, 849, 803, 890, 841, 832, 830, 845, 871, 837, 883 or 809 or functional variants thereof

Also provided is a method for identifying a candidate enzyme having alpha exo rhamnosidase activity, comprising selecting an enzyme comprising, such as consisting of, an amino acid sequence according to SEQ ID No. 992, 1003, 1052, 1073, 1017, 1055, 1075, 1001, 1007, 1061, 1079, 1027, 1039, 1041, 989, 1053, 1018, 1066, 1082, 1076, 993, 1077, 1046, 1015, 1063, 1054, 1074, 1067 or 1033, or functional variants thereof.

Also provided are engineered glucosidase and rhamnosidase polypeptides as further detailed below (referred to as polypeptides of the invention).

The present invention provides an engineered glucosidase polypeptide comprising, such as consisting of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID No. 262, or a functional fragment thereof, wherein the engineered glucosidase polypeptide includes at least one residue substitution from:

-   -   F44Y;     -   V60L;     -   G117A;     -   F170N;     -   V263G or V263L;     -   N351H or N351Q;     -   A355H, A355I, A355L, A355M, A355R, A355T or A355W;     -   A356P;     -   R357A, R357C, R357K, R357M or R357Q;     -   G362C;     -   T365A, T365N or T365S;     -   L367C;     -   V394R;     -   V395Y;     -   Q396E, Q396G, Q396N, Q396P, Q396R, Q396S or Q396Y;     -   F430W;     -   R435F;     -   V438T;     -   V440F;     -   F442M or F442Q;     -   G444T;     -   A473F or A473R;     -   L474C, L474I or L474V;     -   I475F;     -   L492C, L492G, L492H, L492I, L492N, L492Q, L492V, L492W or L492Y;     -   Q493F or Q493H;     -   P494H or P494I;     -   S495I, S495K or S495Q;     -   G496P or G496W;     -   D498A, D498E, D498F, D498I, D498K, D498L, D498N, D498P, D498R,         D498S, D498T or D498V;     -   A502R;     -   M504G or M504R;     -   L507A or L507R;     -   T508M;     -   L529M;     -   F535P;     -   A536D or A536E;     -   A537R;     -   F541A, F541I, F541L, F541M or F541V;     -   L542I;     -   Q543G or Q543L;     -   E547L; and     -   Y585W.

The present invention provides an engineered rhamnosidase polypeptide comprising, such as consisting of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID No. 1017, or a functional fragment thereof, wherein the engineered rhamnosidase polypeptide includes at least one residue substitution from:

-   -   (i) A56C     -   (ii) A143P     -   (iii) Q181H, Q181R or Q181S     -   (iv) L214M     -   (v) G215S     -   (vi) F216M     -   (vii) G218D or G218N     -   (viii) K219G     -   (ix) A238M     -   (x) T252Y     -   (xi) T311W     -   (xii) V326C     -   (xiii) G357C     -   (xiv) S369C, S369I, S369K or S369M     -   (xv) I487M, I487Q or I487V     -   (xvi) K492N     -   (xvii) V499T     -   (xviii) G508S     -   (xix) R543C     -   (xx) L557Y     -   (xxi) G634A     -   (xxii) S635N     -   (xxiii) A690C and     -   (xxiv) Q921H.

The invention also provides a saponin prepared by the methods herein, a saponin containing composition comprising a product saponin prepared by the methods herein, adjuvant compositions comprising said saponins or saponin containing compositions, and adjuvant compositions prepared using said saponins or saponin containing compositions. Use of a saponin or saponin containing composition of the invention in the manufacture of an adjuvant composition is also provided.

Further provided are immunogenic compositions comprising a saponin or saponin containing composition according to the invention and an antigen or a polynucleotide encoding an antigen. Kits comprising (i) a saponin or saponin containing composition according to the invention and (ii) an antigen or a polynucleotide encoding an antigen are also provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 HPLC chromatogram of a crude aqueous Quillaja saponaria bark extract

FIG. 2 HPLC-UV chromatogram of a crude aqueous Quillaja saponaria bark extract

FIG. 3 UPLC-UV chromatogram of a crude aqueous Quillaja saponaria bark extract

FIG. 4 UPLC-UV chromatogram of a polystyrene purified Quillaja saponaria QS-21 saponin extract with low content of 2018 component

FIG. 5 UPLC-UV/MS chromatogram of a Quillaja saponaria QS-21 purified saponin extract with low content of 2018 component

FIG. 6 UPLC-UV/MS chromatogram detail of a Quillaja saponaria QS-21 purified saponin extract with low content of 2018 component

FIG. 7A-7B Extracted mass chromatograms for 1988 (FIG. 7A) and 2002 (FIG. 7B) molecular weight ions of a Quillaja saponaria QS-21 purified saponin extract with low content of 2018 component

FIG. 8 Combined centroid spectrum of Quillaja saponaria QS-21 purified saponin extract with low content of 2018 component

FIG. 9 UPLC-UV chromatogram of Quillaja saponaria QS-21 purified saponin extract with low 2018 component

FIG. 10 LCMS/MS chromatogram for QS-18 2150 (Panel A) and QS-21 1988 (Panel B) component content in QS-21 standard in Example 4-2

FIG. 11 LCMS/MS chromatogram for QS-18 2150 (Panel A) and QS-21 1988 (Panel B) component content following negative control treatment in Example 4-2

FIG. 12 LCMS/MS chromatogram for QS-18 2150 (Panel A) and QS-21 1988 (Panel B) component content following glucosidase SEQ ID No. 262 treatment in Example 4-2

FIG. 13 UV HPLC chromatogram following glucosidase SEQ ID No. 262 treatment (upper trace) and negative control treatment (lower trace) of Crude Bark Extract (CBE) in Example 4-4

FIG. 14 LCMS/MS chromatogram for QS-17 2296 (Panel A) and QS-18 2150 (Panel B) component content following negative control treatment in Example 6-1

FIG. 15 LCMS/MS chromatogram for desglucosyl-QS-17 2134 (Panel A) and QS-21 1988 (Panel B) component content following negative control treatment in Example 6-1

FIG. 16 LCMS/MS chromatogram for QS-17 2310 (Panel A) and QS-18 2164 (Panel B) component content following negative control treatment in Example 6-1

FIG. 17 LCMS/MS chromatogram for QS-17 2296 (Panel A) and QS-18 2150 (Panel B) component content following rhamnosidase SEQ ID No. 1017 treatment in Example 6-1

FIG. 18 LCMS/MS chromatogram for desglucosyl-QS-17 2134 (Panel A) and QS-21 1988 (Panel B) component content following rhamnosidase SEQ ID No. 1017 treatment in Example 6-1

FIG. 19 LCMS/MS chromatogram for QS-17 2310 (Panel A) and QS-18 2164 (Panel B) component content following rhamnosidase SEQ ID No. 1017 treatment in Example 6-1

FIG. 20 UV HPLC chromatogram following rhamnosidase SEQ ID No. 1017 treatment (upper trace) and negative control treatment (lower trace) of Treated Bark Extract (TBE) in Example 6-2

FIG. 21 UV HPLC chromatogram following rhamnosidase SEQ ID No. 1017 treatment (upper trace) and negative control treatment (lower trace) of CBE in Example 6-3

FIG. 22 LCMS/MS chromatogram for QS-21 1988 component content at TO (Panel A) and at 24 hrs (Panel B) following dual enzyme treatment of CBE in Example 7

FIG. 23A-B Illustrative UV HPLC chromatogram following glucosidase enzyme treatment of CBE (FIG. 23 a ) and negative control treatment of CBE (FIG. 23 b ) in Example 8

FIG. 24 Illustrative UV HPLC chromatogram following rhamnosidase enzyme treatment of CBE (upper trace) and negative control treatment of CBE (lower trace) in Example 9

FIG. 25 HPLC-UV chromatogram of untreated and enzyme treated CBE at 1 L scale from Example 11

FIG. 26 UPLC-UV chromatogram following purification of untreated and enzyme treated CBE at 1 L scale from Example 11 (full acquisition)

FIG. 27 UPLC-UV chromatogram following purification of untreated and enzyme treated CBE at 1 L scale from Example 11 (zoom)

BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFIERS

-   -   Seq ID No. 1: Amino acid sequence for Cyberlindnera fabianii         glucosidase, Uniparc reference UPI00049B1A8C, Uniprot reference         A0A061B3J2.     -   Seq ID No. 2: Amino acid sequence for Flavobacterium gilvum         glucosidase, Uniparc reference UPI0004E3EF7B, Uniprot reference         A0A085EII0.     -   Seq ID No. 3: Amino acid sequence for Algibacter lectus         glucosidase, Uniparc reference UPI00050EE490, Uniprot reference         A0A090X649.     -   Seq ID No. 4: Amino acid sequence for Microbacterium         azadirachtae glucosidase, Uniparc reference UPI0005ECB51E,         Uniprot reference A0A0F0LB94.     -   Seq ID No. 5: Amino acid sequence for Actinobacteria bacterium         glucosidase, Uniparc reference UPI0006588DAD, Uniprot reference         A0A0J0UT37.     -   Seq ID No. 6: Amino acid sequence for Chloroflexi bacterium         glucosidase, Uniparc reference UPI0007968552, Uniprot reference         A0A136KWB3.     -   Seq ID No. 7: Amino acid sequence for Komagataeibacter rhaeticus         glucosidase, Uniparc reference UPI0002080410, Uniprot reference         A0A181C809.     -   Seq ID No. 8: Amino acid sequence for Bacteroides sp.         glucosidase, Uniparc reference UPI0008211BFC, Uniprot reference         A0A1C5WEL8.     -   Seq ID No. 9: Amino acid sequence for Streptomyces         rubrolavendulae glucosidase, Uniparc reference UPI00085A2BD0,         Uniprot reference A0A1D8FZW3.     -   Seq ID No. 10: Amino acid sequence for Clostridium roseum         glucosidase, Uniparc reference UPI00098C60F6, Uniprot reference         A0A1S8KYM5.     -   Seq ID No. 11: Amino acid sequence for uncultured bacterium         glucosidase, Uniparc reference UPI0009CE0D4C, Uniprot reference         A0A1V5M6V6.     -   Seq ID No. 12: Amino acid sequence for Firmicutes bacterium         glucosidase, Uniparc reference UPI0009D4127D, Uniprot reference         A0A1V6AN95.     -   Seq ID No. 13: Amino acid sequence for Anthracocystis flocculosa         glucosidase, Uniparc reference UPI00045601AB, Uniprot reference         A0A061H1Z3.     -   Seq ID No. 14: Amino acid sequence for Bifidobacterium boum         glucosidase, Uniparc reference UPI0004FF77C7, Uniprot reference         A0A086ZKU2.     -   Seq ID No. 15: Amino acid sequence for Jejuia pallidilutea         glucosidase, Uniparc reference UPI00051EDBDE, Uniprot reference         A0A098LTR2.     -   Seq ID No. 16: Amino acid sequence for Ceratocystis fimbriata         glucosidase, Uniparc reference UPI00062105AB, Uniprot reference         A0A0F8B2B0.     -   Seq ID No. 17: Amino acid sequence for Actinobacteria bacterium         glucosidase, Uniparc reference UPI0006583AB1, Uniprot reference         A0A0J0UVW7.     -   Seq ID No. 18: Amino acid sequence for Rhodococcus sp.         glucosidase, Uniparc reference UPI0007AABFAD, Uniprot reference         A0A143QAX3.     -   Seq ID No. 19: Amino acid sequence for Valsa mali glucosidase,         Uniparc reference UPI0007F2D02D, Uniprot reference A0A194VF47.     -   Seq ID No. 20: Amino acid sequence for uncultured Bacteroides         sp. glucosidase, Uniparc reference UPI000821004D, Uniprot         reference A0A1C5WSI4.     -   Seq ID No. 21: Amino acid sequence for Eisenbergiella tayi         glucosidase, Uniparc reference UPI00084089B2, Uniprot reference         A0A1E3ALT2.     -   Seq ID No. 22: Amino acid sequence for Streptomyces sp.         glucosidase, Uniparc reference UPI000978E914, Uniprot reference         A0A1V2MY14.     -   Seq ID No. 23: Amino acid sequence for Firmicutes bacterium         glucosidase, Uniparc reference UPI0009CBF21C, Uniprot reference         A0A1V5MH90.     -   Seq ID No. 24: Amino acid sequence for Tenericutes bacterium         glucosidase, Uniparc reference UPI0009D5B1F0, Uniprot reference         A0A1V6BAK3.     -   Seq ID No. 25: Amino acid sequence for Gluconobacter oxydans         glucosidase, Uniparc reference UPI0004A87350, Uniprot reference         A0A067Z479.     -   Seq ID No. 26: Amino acid sequence for Bifidobacterium         catenulatum glucosidase, Uniparc reference UPI00050787A2,         Uniprot reference A0A087B8Q8.     -   Seq ID No. 27: Amino acid sequence for Bionectria ochroleuca         glucosidase, Uniparc reference UPI0005965863, Uniprot reference         A0A0B7K538.     -   Seq ID No. 28: Amino acid sequence for Parcubacteria sp.         glucosidase, Uniparc reference UPI0006377CA4, Uniprot reference         A0A0G0GD78.     -   Seq ID No. 29: Amino acid sequence for Microbacterium         ketosireducens glucosidase, Uniparc reference UPI0006228575,         Uniprot reference A0A0M2H276.     -   Seq ID No. 30: Amino acid sequence for Roseburia faecis         glucosidase, Uniparc reference UPI0006C454B4, Uniprot reference         A0A173R3W4.     -   Seq ID No. 31: Amino acid sequence for Kwoniella dejecticola         glucosidase, Uniparc reference UPI0007F1D695, Uniprot reference         A0A1A6A050.     -   Seq ID No. 32: Amino acid sequence for uncultured Clostridium         sp. glucosidase, Uniparc reference UPI0008232A70, Uniprot         reference A0A1C6C862.     -   Seq ID No. 33: Amino acid sequence for Cyberlindnera jadinii         glucosidase, Uniparc reference UPI000866CB30, Uniprot reference         A0A1E4S2F8.     -   Seq ID No. 34: Amino acid sequence for Bacteroidetes bacterium         glucosidase, Uniparc reference UPI0009D3483D, Uniprot reference         A0A1V5G4W6.     -   Seq ID No. 35: Amino acid sequence for Verrucomicrobia bacterium         glucosidase, Uniparc reference UPI0009CC9AEA, Uniprot reference         A0A1V5Q4R4.     -   Seq ID No. 36: Amino acid sequence for Bacteroidetes bacterium         glucosidase, Uniparc reference UPI0009C6DE73, Uniprot reference         A0A1V6BV25.     -   Seq ID No. 37: Amino acid sequence for Lichtheimia ramosa         glucosidase, Uniparc reference UPI0004E051A9, Uniprot reference         A0A077WUK7.     -   Seq ID No. 38: Amino acid sequence for Bifidobacterium         mongoliense glucosidase, Uniparc reference UPI0005060F52,         Uniprot reference A0A087BWT7.     -   Seq ID No. 39: Amino acid sequence for Vibrio ishigakensis         glucosidase, Uniparc reference UPI0005910ED9, Uniprot reference         A0A0B8NZY1.     -   Seq ID No. 40: Amino acid sequence for Phaeomoniella         chlamydospora glucosidase, Uniparc reference UPI00063B706F,         Uniprot reference A0A0G2HEV5.     -   Seq ID No. 41: Amino acid sequence for Ardenticatena maritima         glucosidase, Uniparc reference UPI0006C04F59, Uniprot reference         A0A0M8K5H7.     -   Seq ID No. 42: Amino acid sequence for Coprococcus comes         glucosidase, Uniparc reference UPI000197E031, Uniprot reference         A0A173WPC4.     -   Seq ID No. 43: Amino acid sequence for Nocardioides dokdonensis         glucosidase, Uniparc reference UPI0007DDBAB3, Uniprot reference         A0A1A9GNJ0.     -   Seq ID No. 44: Amino acid sequence for uncultured Clostridium         sp. glucosidase, Uniparc reference UPI000822F7EB, Uniprot         reference A0A1C6EBC1.     -   Seq ID No. 45: Amino acid sequence for Acetobacterium wieringae         glucosidase, Uniparc reference UPI000878EB40, Uniprot reference         A0A1F2PFB4.     -   Seq ID No. 46: Amino acid sequence for Tenericutes bacterium         glucosidase, Uniparc reference UPI0009D0A2C1, Uniprot reference         A0A1V5HNJ3.     -   Seq ID No. 47: Amino acid sequence for Tenericutes bacterium         glucosidase, Uniparc reference UPI0009CB8A73, Uniprot reference         A0A1V5UFI1.     -   Seq ID No. 48: Amino acid sequence for uncultured bacterium         glucosidase, Uniparc reference UPI0009CC90AF, Uniprot reference         A0A1V6CDT2.     -   Seq ID No. 49: Amino acid sequence for Parabacteroides         distasonis glucosidase, Uniparc reference UPI0004D8E473, Uniprot         reference A0A078SYD0.     -   Seq ID No. 50: Amino acid sequence for Bifidobacterium         psychraerophilum glucosidase, Uniparc reference UPI0005006C6A,         Uniprot reference A0A087CJB0.     -   Seq ID No. 51: Amino acid sequence for Hebeloma cylindrosporum         glucosidase, Uniparc reference UPI00059A3BA8, Uniprot reference         A0A0C2YJF2.     -   Seq ID No. 52: Amino acid sequence for Brenneria goodwinii         glucosidase, Uniparc reference UPI0006579FEA, Uniprot reference         A0A0G4JRR8.     -   Seq ID No. 53: Amino acid sequence for Aspergillus calidoustus         glucosidase, Uniparc reference UPI00073C9E60, Uniprot reference         A0A0U5GN20.     -   Seq ID No. 54: Amino acid sequence for Bacteroides finegoldii         glucosidase, Uniparc reference UPI0006C6E0C3, Uniprot reference         A0A174BEZ2.     -   Seq ID No. 55: Amino acid sequence for Altererythrobacter         dongtanensis glucosidase, Uniparc reference UPI0008153D6D,         Uniprot reference A0A1B2A943.     -   Seq ID No. 56: Amino acid sequence for uncultured Anaerotruncus         sp. glucosidase, Uniparc reference UPI0008206E7B, Uniprot         reference A0A1C6FWD9.     -   Seq ID No. 57: Amino acid sequence for Candidatus firestone         glucosidase, Uniparc reference UPI0008AC0E17, Uniprot reference         A0A1F5UJR6.     -   Seq ID No. 58: Amino acid sequence for Tenericutes bacterium         glucosidase, Uniparc reference UPI0009C6C8E4, Uniprot reference         A0A1V5HS22.     -   Seq ID No. 59: Amino acid sequence for Lentisphaerae bacterium         glucosidase, Uniparc reference UPI0009CEE1AB, Uniprot reference         A0A1V5VBL2.     -   Seq ID No. 60: Amino acid sequence for Planctomycetes bacterium         glucosidase, Uniparc reference UPI0009CAAB0A, Uniprot reference         A0A1V6FZ47.     -   Seq ID No. 61: Amino acid sequence for Pseudallescheria         apiosperma glucosidase, Uniparc reference UPI0004DD62AC, Uniprot         reference A0A084G332.     -   Seq ID No. 62: Amino acid sequence for Nonlabens sediminis         glucosidase, Uniparc reference UPI000507F00A, Uniprot reference         A0A090Q4N8.     -   Seq ID No. 63: Amino acid sequence for Gynuella sunshinyii         glucosidase, Uniparc reference UPI0005CC42CA, Uniprot reference         A0A0C5VDU3.     -   Seq ID No. 64: Amino acid sequence for Verticillium longisporum         glucosidase, Uniparc reference UPI00063E4005, Uniprot reference         A0A0G4N9Q7.     -   Seq ID No. 65: Amino acid sequence for Cellulomonas sp.         glucosidase, Uniparc reference UPI00073C6CD3, Uniprot reference         A0A0V8TAB5.     -   Seq ID No. 66: Amino acid sequence for Hungatella hathewayi         glucosidase, Uniparc reference UPI0006C024BB, Uniprot reference         A0A174FBX7.     -   Seq ID No. 67: Amino acid sequence for Mesorhizobium sp.         glucosidase, Uniparc reference UPI000688E74D, Uniprot reference         A0A1C2DG64.     -   Seq ID No. 68: Amino acid sequence for Clostridium sp.         glucosidase, Uniparc reference UPI000822FAB7, Uniprot reference         A0A1C6GRT5.     -   Seq ID No. 69: Amino acid sequence for Chlamydiales bacterium         glucosidase, Uniparc reference UPI0009284E74, Uniprot reference         A0A1M3CSY6.     -   Seq ID No. 70: Amino acid sequence for Spirochaetes bacterium         glucosidase, Uniparc reference UPI0009CB461D, Uniprot reference         A0A1V5HUX3.     -   Seq ID No. 71: Amino acid sequence for bacterium glucosidase,         Uniparc reference UPI0009C92D6A, Uniprot reference A0A1V5VHB9.     -   Seq ID No. 72: Amino acid sequence for Thermotogae bacterium         glucosidase, Uniparc reference UPI0009D12B86, Uniprot reference         A0A1V6H2W5.     -   Seq ID No. 73: Amino acid sequence for Pseudallescheria         apiosperma glucosidase, Uniparc reference UPI0004DCBED3, Uniprot         reference A0A084GGE2.     -   Seq ID No. 74: Amino acid sequence for Algibacter lectus         glucosidase, Uniparc reference UPI00050E0BE3, Uniprot reference         A0A090VF17.     -   Seq ID No. 75: Amino acid sequence for Paxillus involutus         glucosidase, Uniparc reference UPI0005B075C8, Uniprot reference         A0A0C9TWP5.     -   Seq ID No. 76: Amino acid sequence for Verticillium longisporum         glucosidase, Uniparc reference UPI00063DF006, Uniprot reference         A0A0G4NA55.     -   Seq ID No. 77: Amino acid sequence for Mucilaginibacter         gotjawali glucosidase, Uniparc reference UPI00076F8EA4, Uniprot         reference A0A110B1H1.     -   Seq ID No. 78: Amino acid sequence for Bacteroides uniformis         glucosidase, Uniparc reference UPI0006C0FFEF, Uniprot reference         A0A174IWW4.     -   Seq ID No. 79: Amino acid sequence for Coprococcus sp.         glucosidase, Uniparc reference UPI0008222B77, Uniprot reference         A0A1C5W0N6.     -   Seq ID No. 80: Amino acid sequence for Blautia sp. glucosidase,         Uniparc reference UPI0006C36823, Uniprot reference A0A1C6K2X5.     -   Seq ID No. 81: Amino acid sequence for Cellulomonas sp.         glucosidase, Uniparc reference UPI00092B3CD4, Uniprot reference         A0A1M3ELH4.     -   Seq ID No. 82: Amino acid sequence for bacterium glucosidase,         Uniparc reference UPI0009C85428, Uniprot reference A0A1V5J984.     -   Seq ID No. 83: Amino acid sequence for Spirochaetes bacterium         glucosidase, Uniparc reference UPI0009CA1D5C, Uniprot reference         A0A1V5WHQ7.     -   Seq ID No. 84: Amino acid sequence for Tenericutes bacterium         glucosidase, Uniparc reference UPI0009C7041F, Uniprot reference         A0A1V6IJ57.     -   Seq ID No. 85: Amino acid sequence for Flavobacterium gilvum         glucosidase, Uniparc reference UPI0004E2A41F, Uniprot reference         A0A085EG29.     -   Seq ID No. 86: Amino acid sequence for Algibacter lectus         glucosidase, Uniparc reference UPI0005102470, Uniprot reference         A0A090WWZ1.     -   Seq ID No. 87: Amino acid sequence for Hydnomerulius pinastri         glucosidase, Uniparc reference UPI0005B0F0AE, Uniprot reference         A0A0C9WDY0.     -   Seq ID No. 88: Amino acid sequence for Nocardia farcinica         glucosidase, Uniparc reference UPI00065C2666, Uniprot reference         A0A0H5NWN2.     -   Seq ID No. 89: Amino acid sequence for Bacteroides         cellulosilyticus glucosidase, Uniparc reference UPI000760375B,         Uniprot reference A0A125MG18.     -   Seq ID No. 90: Amino acid sequence for Fonsecaea erecta         glucosidase, Uniparc reference UPI0007DF4250, Uniprot reference         A0A178ZBN2.     -   Seq ID No. 91: Amino acid sequence for Bacteroides sp.         glucosidase, Uniparc reference UPI0008209852, Uniprot reference         A0A1C5W9N9.     -   Seq ID No. 92: Amino acid sequence for Tannerella forsythia         glucosidase, Uniparc reference UPI00086C34C1, Uniprot reference         A0A1D3UGH8.     -   Seq ID No. 93: Amino acid sequence for Microbacterium         esteraromaticum glucosidase, Uniparc reference UPI00097E83BB,         Uniprot reference A0A1R4KI92.     -   Seq ID No. 94: Amino acid sequence for bacterium glucosidase,         Uniparc reference UPI0009D255E5, Uniprot reference A0A1V5LJK9.     -   Seq ID No. 95: Amino acid sequence for Candidatus         hydrogenedentes glucosidase, Uniparc reference UPI0009C5A3CF,         Uniprot reference A0A1V5Z2L2.     -   Seq ID No. 96: Amino acid sequence for Bacteroidetes bacterium         glucosidase, Uniparc reference UPI0009C55799, Uniprot reference         A0A1V6J4J8.     -   Seq ID No. 97: Amino acid sequence for Penicillium solitum         glucosidase, Uniparc reference UPI0009D4067F, Uniprot reference         A0A1V6RQ41.     -   Seq ID No. 98: Amino acid sequence for Weissella soli         glucosidase, Uniparc reference UPI0008737AA2, Uniprot reference         A0A288Q8I2.     -   Seq ID No. 99: Amino acid sequence for Acetatifactor muris         glucosidase, Uniparc reference UPI000CAC57D4, Uniprot reference         A0A2K4ZN91.     -   Seq ID No. 100: Amino acid sequence for Corynespora cassiicola         glucosidase, Uniparc reference UPI000D237A4A, Uniprot reference         A0A2T2NYD4.     -   Seq ID No. 101: Amino acid sequence for Meira miltonrushii         glucosidase, Uniparc reference UPI000D77C91D, Uniprot reference         A0A316V6M3.     -   Seq ID No. 102: Amino acid sequence for Bacteroides fragilis         glucosidase, Uniparc reference UPI00004E1F76, Uniprot reference         A0A380YVC7.     -   Seq ID No. 103: Amino acid sequence for Malassezia restricta         glucosidase, Uniparc reference UPI000F0C30E8, Uniprot reference         A0A3G2SB79.     -   Seq ID No. 104: Amino acid sequence for Fusarium euwallaceae         glucosidase, Uniparc reference UPI000FFFEFB2, Uniprot reference         A0A430LYA2.     -   Seq ID No. 105: Amino acid sequence for Psathyrella aberdarensis         glucosidase, Uniparc reference UPI0010251887, Uniprot reference         A0A4Q2E070.     -   Seq ID No. 106: Amino acid sequence for Aeromonas hydrophila         glucosidase, Uniparc reference UPI0000E69509, Uniprot reference         A0KLP6.     -   Seq ID No. 107: Amino acid sequence for Saccharopolyspora         erythraea glucosidase, Uniparc reference UPI00000B86CB, Uniprot         reference A4F7P9.     -   Seq ID No. 108: Amino acid sequence for Streptomyces sviceus         glucosidase, Uniparc reference UPI000180240E, Uniprot reference         B5I181.     -   Seq ID No. 109: Amino acid sequence for Naematelia encephala         glucosidase, Uniparc reference UPI000A250F78, Uniprot reference         A0A1Y2AWB7.     -   Seq ID No. 110: Amino acid sequence for Hartmannibacter         diazotrophicus glucosidase, Uniparc reference UPI000C0221F1,         Uniprot reference A0A2C9D6I2.     -   Seq ID No. 111: Amino acid sequence for Pontimonas salivibrio         glucosidase, Uniparc reference UPI000CEB5AB1, Uniprot reference         A0A2L2BPE2.     -   Seq ID No. 112: Amino acid sequence for Cadophora sp.         glucosidase, Uniparc reference UPI000D5B9C38, Uniprot reference         A0A2V1CH24.     -   Seq ID No. 113: Amino acid sequence for Meira miltonrushii         glucosidase, Uniparc reference UPI000D779558, Uniprot reference         A0A316V8S0.     -   Seq ID No. 114: Amino acid sequence for Monilinia fructigena         glucosidase, Uniparc reference UPI000DC42E3D, Uniprot reference         A0A395IJW4.     -   Seq ID No. 115: Amino acid sequence for Hortaea werneckii         glucosidase, Uniparc reference UPI000F3E476C, Uniprot reference         A0A3M6XGS0.     -   Seq ID No. 116: Amino acid sequence for Streptomyces netropsis         glucosidase, Uniparc reference UPI00101460D7, Uniprot reference         A0A445N7U0.     -   Seq ID No. 117: Amino acid sequence for Aureobasidium pullulans         glucosidase, Uniparc reference UPI001139C6A8, Uniprot reference         A0A4S9IFI0.     -   Seq ID No. 118: Amino acid sequence for Aspergillus clavatus         glucosidase, Uniparc reference UPI0000EA5CFF, Uniprot reference         A1CTN9.     -   Seq ID No. 119: Amino acid sequence for Clavibacter         michiganensis glucosidase, Uniparc reference UPI0001523037,         Uniprot reference A5CT94.     -   Seq ID No. 120: Amino acid sequence for Penicillium rubens         glucosidase, Uniparc reference UPI0001831CF5, Uniprot reference         B6H7R5.     -   Seq ID No. 121: Amino acid sequence for Lachnoclostridium sp.         glucosidase, Uniparc reference UPI000B365547, Uniprot reference         A0A1Y4NTL9.     -   Seq ID No. 122: Amino acid sequence for Rhodobacteraceae         bacterium glucosidase, Uniparc reference UPI000C09BF88, Uniprot         reference A0A2D5IXB9.     -   Seq ID No. 123: Amino acid sequence for Bacteroides fragilis         glucosidase, Uniparc reference UPI0004B5EEF2, Uniprot reference         A0A2M9UUC4.     -   Seq ID No. 124: Amino acid sequence for Aspergillus indologenus         glucosidase, Uniparc reference UPI000D7FE1ED, Uniprot reference         A0A2V5IY78.     -   Seq ID No. 125: Amino acid sequence for Acaromyces ingoldii         glucosidase, Uniparc reference UPI000D802B25, Uniprot reference         A0A316YR39.     -   Seq ID No. 126: Amino acid sequence for Monilinia fructigena         glucosidase, Uniparc reference UPI000DC60823, Uniprot reference         A0A395J1U5.     -   Seq ID No. 127: Amino acid sequence for Paenibacillus         xylanexedens glucosidase, Uniparc reference UPI000F52D773,         Uniprot reference A0A3N6CA02.     -   Seq ID No. 128: Amino acid sequence for Actinomyces howellii         glucosidase, Uniparc reference UPI000F6DAAAE, Uniprot reference         A0A448HIG0.     -   Seq ID No. 129: Amino acid sequence for Friedmanniomyces         endolithicus glucosidase, Uniparc reference UPI00113D19DF,         Uniprot reference A0A4V5N9I4.     -   Seq ID No. 130: Amino acid sequence for Neosartorya fischeri         glucosidase, Uniparc reference UPI0000EA8672, Uniprot reference         A1DNS0.     -   Seq ID No. 131: Amino acid sequence for Pseudomonas aeruginosa         glucosidase, Uniparc reference UPI0000D7314B, Uniprot reference         A6V4K6.     -   Seq ID No. 132: Amino acid sequence for Talaromyces stipitatus         glucosidase, Uniparc reference UPI00018E7266, Uniprot reference         B8MF24.     -   Seq ID No. 133: Amino acid sequence for Aquimixticola         soesokkakensis glucosidase, Uniparc reference UPI000A1A5FD7,         Uniprot reference A0A1Y5RVF8.     -   Seq ID No. 134: Amino acid sequence for Rhodobacterales         bacterium glucosidase, Uniparc reference UPI000C98D37C, Uniprot         reference A0A2D9YGV1.     -   Seq ID No. 135: Amino acid sequence for Methylorubrum extorquens         glucosidase, Uniparc reference UPI0006F9793E, Uniprot reference         A0A2N9AS40.     -   Seq ID No. 136: Amino acid sequence for Clostridium perfringens         glucosidase, Uniparc reference UPI00000CF7C8, Uniprot reference         A0A2X2YBP0.     -   Seq ID No. 137: Amino acid sequence for Acholeplasmatales         bacterium glucosidase, Uniparc reference UPI0008AEBEA3, Uniprot         reference A0A348NID6.     -   Seq ID No. 138: Amino acid sequence for Bacteroidetes bacterium         glucosidase, Uniparc reference UPI000EC3C979, Uniprot reference         A0A3B8VHE8.     -   Seq ID No. 139: Amino acid sequence for Clostridium carnis         glucosidase, Uniparc reference UPI000F637E38, Uniprot reference         A0A3P6K8E8.     -   Seq ID No. 140: Amino acid sequence for Mycolicibacterium         flavescens glucosidase, Uniparc reference UPI000B93B5C9, Uniprot         reference A0A448HNB5.     -   Seq ID No. 141: Amino acid sequence for Streptococcus         gallolyticus glucosidase, Uniparc reference UPI000F6EFA71,         Uniprot reference A0A4V6LJ94.     -   Seq ID No. 142: Amino acid sequence for Yersinia enterocolitica         glucosidase, Uniparc reference UPI0000EB54CC, Uniprot reference         A1JNB7.     -   Seq ID No. 143: Amino acid sequence for Anaeromyxobacter sp.         glucosidase, Uniparc reference UPI0000ED8A80, Uniprot reference         A7HFG4.     -   Seq ID No. 144: Amino acid sequence for Talaromyces stipitatus         glucosidase, Uniparc reference UPI00018E7D70, Uniprot reference         B8MK55.     -   Seq ID No. 145: Amino acid sequence for Hortaea werneckii         glucosidase, Uniparc reference UPI000A2E3FAA, Uniprot reference         A0A1Z5SL14.     -   Seq ID No. 146: Amino acid sequence for Micavibrio sp.         glucosidase, Uniparc reference UPI000C529025, Uniprot reference         A0A2E2Q8X2.     -   Seq ID No. 147: Amino acid sequence for Acidobacteriia bacterium         glucosidase, Uniparc reference UPI000CE6B996, Uniprot reference         A0A2N9MBS0.     -   Seq ID No. 148: Amino acid sequence for Corynebacterium jeikeium         glucosidase, Uniparc reference UPI000DA3A972, Uniprot reference         A0A2X4T570.     -   Seq ID No. 149: Amino acid sequence for Clostridiaceae bacterium         glucosidase, Uniparc reference UPI000E8D37A1, Uniprot reference         A0A353PZH8.     -   Seq ID No. 150: Amino acid sequence for Anaerolineaceae         bacterium glucosidase, Uniparc reference UPI000748C096, Uniprot         reference A0A3B9PA35.     -   Seq ID No. 151: Amino acid sequence for Gymnopilus dilepis         glucosidase, Uniparc reference UPI000FF41956, Uniprot reference         A0A409WSY0.     -   Seq ID No. 152: Amino acid sequence for Kocuria rosea         glucosidase, Uniparc reference UPI000F7105D4, Uniprot reference         A0A448R8N0.     -   Seq ID No. 153: Amino acid sequence for Teredinibacter sp.         glucosidase, Uniparc reference UPI0011696FAB, Uniprot reference         A0A509DWZ3.     -   Seq ID No. 154: Amino acid sequence for Aspergillus niger         glucosidase, Uniparc reference UPI0000EFB564, Uniprot reference         A2QS42.     -   Seq ID No. 155: Amino acid sequence for Laccaria bicolor         glucosidase, Uniparc reference UPI000164423D, Uniprot reference         B0D734.     -   Seq ID No. 156: Amino acid sequence for Podosphaera parvula         glucosidase, Uniparc reference UPI00017357F6, Uniprot reference         B9XH33.     -   Seq ID No. 157: Amino acid sequence for Megamonas hypermegale         glucosidase, Uniparc reference UPI00042469F6, Uniprot reference         A0A239TGH2.     -   Seq ID No. 158: Amino acid sequence for Armillaria gallica         glucosidase, Uniparc reference UPI000BC209C3, Uniprot reference         A0A2H3E300.     -   Seq ID No. 159: Amino acid sequence for Micromonospora sp.         glucosidase, Uniparc reference UPI000D2EAE87, Uniprot reference         A0A2P8AV03.     -   Seq ID No. 160: Amino acid sequence for Klebsiella oxytoca         glucosidase, Uniparc reference UPI000DA286EE, Uniprot reference         A0A2X5CJC5.     -   Seq ID No. 161: Amino acid sequence for Candidatus ozemobacter         glucosidase, Uniparc reference UPI000DFAEF6C, Uniprot reference         A0A367ZIH8.     -   Seq ID No. 162: Amino acid sequence for Coleophoma crateriformis         glucosidase, Uniparc reference UPI000E399EE8, Uniprot reference         A0A3D8R2C2.     -   Seq ID No. 163: Amino acid sequence for Apiotrichum porosum         glucosidase, Uniparc reference UPI000FBC01E3, Uniprot reference         A0A427XHS2.     -   Seq ID No. 164: Amino acid sequence for Acholeplasma hippikon         glucosidase, Uniparc reference UPI00068E4E50, Uniprot reference         A0A449BJ27.     -   Seq ID No. 165: Amino acid sequence for Streptomyces spectabilis         glucosidase, Uniparc reference UPI001185F074, Uniprot reference         A0A516RGT1.     -   Seq ID No. 166: Amino acid sequence for Aspergillus niger         glucosidase, Uniparc reference UPI0000EFCED2, Uniprot reference         A2R8G2.     -   Seq ID No. 167: Amino acid sequence for Xanthomonas campestris         glucosidase, Uniparc reference UPI00000D8BFA, Uniprot reference         B0RYA0.     -   Seq ID No. 168: Amino acid sequence for Lactobacillus paracasei         glucosidase, Uniparc reference UPI00019C9CD7, Uniprot reference         C2FDL2.     -   Seq ID No. 169: Amino acid sequence for Bifiguratus adelaidae         glucosidase, Uniparc reference UPI000BC490A3, Uniprot reference         A0A261XUH4.     -   Seq ID No. 170: Amino acid sequence for bacterium glucosidase,         Uniparc reference UPI000CC31AE2, Uniprot reference A0A2H5YYA1.     -   Seq ID No. 171: Amino acid sequence for Actinomadura parvosata         glucosidase, Uniparc reference UPI000D26C9C6, Uniprot reference         A0A2P9IY35.     -   Seq ID No. 172: Amino acid sequence for Melissococcus plutonius         glucosidase, Uniparc reference UPI00024F22B8, Uniprot reference         A0A2Z5Y4P3.     -   Seq ID No. 173: Amino acid sequence for Enterococcus durans         glucosidase, Uniparc reference UPI000E020871, Uniprot reference         A0A377KJS3.     -   Seq ID No. 174: Amino acid sequence for Malassezia restricta         glucosidase, Uniparc reference UPI000DD17A7D, Uniprot reference         A0A3G2S2J6.     -   Seq ID No. 175: Amino acid sequence for Apiotrichum porosum         glucosidase, Uniparc reference UPI000FA2DE87, Uniprot reference         A0A427XZQ0.     -   Seq ID No. 176: Amino acid sequence for Streptomonospora sp.         glucosidase, Uniparc reference UPI0010355193, Uniprot reference         A0A4P6PWP5.     -   Seq ID No. 177: Amino acid sequence for Lactobacillus gasseri         glucosidase, Uniparc reference UPI00119640A1, Uniprot reference         A0A558LH47.     -   Seq ID No. 178: Amino acid sequence for Aspergillus niger         glucosidase, Uniparc reference UPI0000EFD0AA, Uniprot reference         A2RAJ1.     -   Seq ID No. 179: Amino acid sequence for Neosartorya fumigata         glucosidase, Uniparc reference UPI000170BF91, Uniprot reference         B0XXG1.     -   Seq ID No. 180: Amino acid sequence for Eubacterium eligens         glucosidase, Uniparc reference UPI0001A5B234, Uniprot reference         C4Z6T5.     -   Seq ID No. 181: Amino acid sequence for Bifiguratus adelaidae         glucosidase, Uniparc reference UPI000BC64A75, Uniprot reference         A0A261XVM8.     -   Seq ID No. 182: Amino acid sequence for bacterium glucosidase,         Uniparc reference UPI000CAA854E, Uniprot reference A0A2H6EX57.     -   Seq ID No. 183: Amino acid sequence for Corynespora cassiicola         glucosidase, Uniparc reference UPI000D24EC2C, Uniprot reference         A0A2T2N5H9.     -   Seq ID No. 184: Amino acid sequence for Pseudomicrostroma         glucosiphilum glucosidase, Uniparc reference UPI000D77A0B7,         Uniprot reference A0A316UK83.     -   Seq ID No. 185: Amino acid sequence for Staphylococcus         saprophyticus glucosidase, Uniparc reference UPI0002DAAFCC,         Uniprot reference A0A380HD31.     -   Seq ID No. 186: Amino acid sequence for Malassezia restricta         glucosidase, Uniparc reference UPI000F0C6B8E, Uniprot reference         A0A3G2S932.     -   Seq ID No. 187: Amino acid sequence for Saitozyma podzolica         glucosidase, Uniparc reference UPI000FBA70E2, Uniprot reference         A0A427YCL1.     -   Seq ID No. 188: Amino acid sequence for Tremella mesenterica         glucosidase, Uniparc reference UPI00102822B4, Uniprot reference         A0A4Q1BRC6.     -   Seq ID No. 189: Amino acid sequence for Arthrobacter sp.         glucosidase, Uniparc reference UPI0000527506, Uniprot reference         A0JZ86.     -   Seq ID No. 190: Amino acid sequence for Scheffersomyces stipitis         glucosidase, Uniparc reference UPI000157388C, Uniprot reference         A3LRB0.     -   Seq ID No. 191: Amino acid sequence for Leptothrix cholodnii         glucosidase, Uniparc reference UPI0001712E02, Uniprot reference         B1XZK8.     -   Seq ID No. 192: Amino acid sequence for Thauera sp. glucosidase,         Uniparc reference UPI000166883C, Uniprot reference C4ZLL7.     -   Seq ID No. 193: Amino acid sequence for Kosmotoga olearia         glucosidase, Uniparc reference UPI00018494AB, Uniprot reference         C5CDW5.     -   Seq ID No. 194: Amino acid sequence for Roseburia intestinalis         glucosidase, Uniparc reference UPI0001CD671F, Uniprot reference         D4L3Y2.     -   Seq ID No. 195: Amino acid sequence for Streptococcus equinus         glucosidase, Uniparc reference UPI0001E0DC00, Uniprot reference         E0PDF8.     -   Seq ID No. 196: Amino acid sequence for Streptococcus cristatus         glucosidase, Uniparc reference UPI0001F80C4B, Uniprot reference         E8JUK5.     -   Seq ID No. 197: Amino acid sequence for Cellulosilyticum         lentocellum glucosidase, Uniparc reference UPI0001D2DBBC,         Uniprot reference F2JLH3.     -   Seq ID No. 198: Amino acid sequence for Streptococcus         gallolyticus glucosidase, Uniparc reference UPI0001C48657,         Uniprot reference F5WYI5.     -   Seq ID No. 199: Amino acid sequence for Ketogulonicigenium         vulgare glucosidase, Uniparc reference UPI00021D443A, Uniprot         reference F9Y8Z7.     -   Seq ID No. 200: Amino acid sequence for Spathaspora passalidarum         glucosidase, Uniparc reference UPI000228255D, Uniprot reference         G3AGX1.     -   Seq ID No. 201: Amino acid sequence for Niastella koreensis         glucosidase, Uniparc reference UPI00023F6F5F, Uniprot reference         G8T9J3.     -   Seq ID No. 202: Amino acid sequence for Cellvibrio sp.         glucosidase, Uniparc reference UPI000260108C, Uniprot reference         I3IDC0.     -   Seq ID No. 203: Amino acid sequence for Flavobacterium sp.         glucosidase, Uniparc reference UPI000272D1E0, Uniprot reference         J1ACA0.     -   Seq ID No. 204: Amino acid sequence for Macrophomina phaseolina         glucosidase, Uniparc reference UPI00028E7FE1, Uniprot reference         K2S5D3.     -   Seq ID No. 205: Amino acid sequence for Kosmotoga olearia         glucosidase, Uniparc reference UPI00018483A2, Uniprot reference         C5CHI5.     -   Seq ID No. 206: Amino acid sequence for Blautia obeum         glucosidase, Uniparc reference UPI0001CD5918, Uniprot reference         D4LRF6.     -   Seq ID No. 207: Amino acid sequence for Bifidobacterium dentium         glucosidase, Uniparc reference UPI0001E18CDA, Uniprot reference         E0Q541.     -   Seq ID No. 208: Amino acid sequence for Anaerolinea thermophila         glucosidase, Uniparc reference UPI0001F55F8F, Uniprot reference         E8N5R8.     -   Seq ID No. 209: Amino acid sequence for Coriobacterium glomerans         glucosidase, Uniparc reference UPI0002050DA2, Uniprot reference         F2N7E4.     -   Seq ID No. 210: Amino acid sequence for Microlunatus         phosphovorus glucosidase, Uniparc reference UPI000210C886,         Uniprot reference F5XJQ3.     -   Seq ID No. 211: Amino acid sequence for Streptomyces sp.         glucosidase, Uniparc reference UPI0001C18877, Uniprot reference         G0Q1U8.     -   Seq ID No. 212: Amino acid sequence for Spathaspora passalidarum         glucosidase, Uniparc reference UPI0002282B61, Uniprot reference         G3AIV6.     -   Seq ID No. 213: Amino acid sequence for Glarea lozoyensis         glucosidase, Uniparc reference UPI0002402F4A, Uniprot reference         H0ET34.     -   Seq ID No. 214: Amino acid sequence for Glaciozyma antarctica         glucosidase, Uniparc reference UPI0002633B36, Uniprot reference         I3UJK0.     -   Seq ID No. 215: Amino acid sequence for Arthrobacter sp.         glucosidase, Uniparc reference UPI00027DFD35, Uniprot reference         J7LN00.     -   Seq ID No. 216: Amino acid sequence for Fusarium         pseudograminearum glucosidase, Uniparc reference UPI00028D698C,         Uniprot reference K3VMA9.     -   Seq ID No. 217: Amino acid sequence for Hypocrea rufa         glucosidase, Uniparc reference UPI000006AA61, Uniprot reference         C6GGC9.     -   Seq ID No. 218: Amino acid sequence for Ruminococcus torques         glucosidase, Uniparc reference UPI0001CDA964, Uniprot reference         D4M6W6.     -   Seq ID No. 219: Amino acid sequence for Bifidobacterium dentium         glucosidase, Uniparc reference UPI0001E17370, Uniprot reference         E0Q9Z7.     -   Seq ID No. 220: Amino acid sequence for Bacteroides salanitronis         glucosidase, Uniparc reference UPI0001FC71F6, Uniprot reference         F0R2D7.     -   Seq ID No. 221: Amino acid sequence for Bacteroides coprosuis         glucosidase, Uniparc reference UPI00020E6D19, Uniprot reference         F3ZQ40.     -   Seq ID No. 222: Amino acid sequence for Marinomonas posidonica         glucosidase, Uniparc reference UPI00020D4CC6, Uniprot reference         F6CWF6.     -   Seq ID No. 223: Amino acid sequence for Chaetomium thermophilum         glucosidase, Uniparc reference UPI000227E8ED, Uniprot reference         G0SE64.     -   Seq ID No. 224: Amino acid sequence for Tetragenococcus         halophilus glucosidase, Uniparc reference UPI00022B9A15, Uniprot         reference G4L5K1.     -   Seq ID No. 225: Amino acid sequence for Paenibacillus sp.         glucosidase, Uniparc reference UPI00024F0867, Uniprot reference         H6CCZ8.     -   Seq ID No. 226: Amino acid sequence for Turneriella parva         glucosidase, Uniparc reference UPI000265AA4B, Uniprot reference         I4B8U7.     -   Seq ID No. 227: Amino acid sequence for Arthrobacter sp.         glucosidase, Uniparc reference UPI00027DFDB2, Uniprot reference         J7LQK9.     -   Seq ID No. 228: Amino acid sequence for Agaricus bisporus         glucosidase, Uniparc reference UPI00029074B1, Uniprot reference         K5W7V1.     -   Seq ID No. 229: Amino acid sequence for Nectria haematococca         glucosidase, Uniparc reference UPI0001B67634, Uniprot reference         C7YIP3.     -   Seq ID No. 230: Amino acid sequence for Bacteroides         xylanisolvens glucosidase, Uniparc reference UPI0001A25287,         Uniprot reference D4VSZ0.     -   Seq ID No. 231: Amino acid sequence for Sediminispirochaeta         smaragdinae glucosidase, Uniparc reference UPI0001DD9790,         Uniprot reference E1R331.     -   Seq ID No. 232: Amino acid sequence for Deinococcus         proteolyticus glucosidase, Uniparc reference UPI0001FC42E9,         Uniprot reference F0RPV2.     -   Seq ID No. 233: Amino acid sequence for Sphingobacterium sp.         glucosidase, Uniparc reference UPI0002033A0A, Uniprot reference         F4C226.     -   Seq ID No. 234: Amino acid sequence for Sphingobium         chlorophenolicum glucosidase, Uniparc reference UPI0001E54133,         Uniprot reference F6ET40.     -   Seq ID No. 235: Amino acid sequence for Caloramator australicus         glucosidase, Uniparc reference UPI00021CACC4, Uniprot reference         G0V3V5.     -   Seq ID No. 236: Amino acid sequence for Commensalibacter         intestini glucosidase, Uniparc reference UPI000230E3BF, Uniprot         reference G6F370.     -   Seq ID No. 237: Amino acid sequence for Paenibacillus sp.         glucosidase, Uniparc reference UPI00024F07AC, Uniprot reference         H6CIT2.     -   Seq ID No. 238: Amino acid sequence for Nitrolancea hollandica         glucosidase, Uniparc reference UPI0002638AF3, Uniprot reference         I4EIA9.     -   Seq ID No. 239: Amino acid sequence for Cryptococcus neoformans         glucosidase, Uniparc reference UPI000392C3ED, Uniprot reference         J9VVK7.     -   Seq ID No. 240: Amino acid sequence for Acidipropionibacterium         acidipropionici glucosidase, Uniparc reference UPI0002988588,         Uniprot reference K75596.     -   Seq ID No. 241: Amino acid sequence for Prevotella sp.         glucosidase, Uniparc reference UPI0001B93465, Uniprot reference         C9PT75.     -   Seq ID No. 242: Amino acid sequence for Rhodobacter capsulatus         glucosidase, Uniparc reference UPI0001D08095, Uniprot reference         D5ALU0.     -   Seq ID No. 243: Amino acid sequence for Stigmatella aurantiaca         glucosidase, Uniparc reference UPI0001E74370, Uniprot reference         E3FJ05.     -   Seq ID No. 244: Amino acid sequence for Sphaerochaeta globosa         glucosidase, Uniparc reference UPI0002010060, Uniprot reference         F0RVK3.     -   Seq ID No. 245: Amino acid sequence for Sphaerochaeta coccoides         glucosidase, Uniparc reference UPI000207D78C, Uniprot reference         F4GH96.     -   Seq ID No. 246: Amino acid sequence for Novosphingobium sp.         glucosidase, Uniparc reference UPI00020EFBCB, Uniprot reference         F6ICQ5.     -   Seq ID No. 247: Amino acid sequence for Arthrobotrys oligospora         glucosidase, Uniparc reference UPI000225331F, Uniprot reference         G1XH86.     -   Seq ID No. 248: Amino acid sequence for Lactococcus lactis         glucosidase, Uniparc reference UPI00000C6907, Uniprot reference         G6FFS4.     -   Seq ID No. 249: Amino acid sequence for Phaeospirillum         molischianum glucosidase, Uniparc reference UPI000255313A,         Uniprot reference H8FXH7.     -   Seq ID No. 250: Amino acid sequence for Modestobacter marinus         glucosidase, Uniparc reference UPI0002609C68, Uniprot reference         I4EW72.     -   Seq ID No. 251: Amino acid sequence for Saccharothrix         espanaensis glucosidase, Uniparc reference UPI00028AC337,         Uniprot reference K0K125.     -   Seq ID No. 252: Amino acid sequence for Cronobacter sakazakii         glucosidase, Uniparc reference UPI00029BA293, Uniprot reference         K8DAK0.     -   Seq ID No. 253: Amino acid sequence for Verticillium alfalfae         glucosidase, Uniparc reference UPI0001BBDF1E, Uniprot reference         C9SVX1.     -   Seq ID No. 254: Amino acid sequence for Bacteroides         xylanisolvens glucosidase, Uniparc reference UPI0001CCFD36,         Uniprot reference D6CY10.     -   Seq ID No. 255: Amino acid sequence for Leadbetterella         byssophila glucosidase, Uniparc reference UPI0001EBD98A, Uniprot         reference E4RUR0.     -   Seq ID No. 256: Amino acid sequence for Sphaerochaeta globosa         glucosidase, Uniparc reference UPI00020102A5, Uniprot reference         F0RYB6.     -   Seq ID No. 257: Amino acid sequence for Sphaerochaeta coccoides         glucosidase, Uniparc reference UPI000207D6A0, Uniprot reference         F4GLH6.     -   Seq ID No. 258: Amino acid sequence for Haloplasma contractile         glucosidase, Uniparc reference UPI000212252C, Uniprot reference         F7Q0Y2.     -   Seq ID No. 259: Amino acid sequence for Nitrospirillum         amazonense glucosidase, Uniparc reference UPI0002265447, Uniprot         reference G1Y4C7.     -   Seq ID No. 260: Amino acid sequence for Azospirillum brasilense         glucosidase, Uniparc reference UPI00023420B8, Uniprot reference         G8AWD9.     -   Seq ID No. 261: Amino acid sequence for Phaeospirillum         molischianum glucosidase, Uniparc reference UPI000255314F,         Uniprot reference H8FXJ8.     -   Seq ID No. 262: Amino acid sequence for Modestobacter marinus         glucosidase, Uniparc reference UPI000260A2FA, Uniprot reference         I4EYD5.     -   Seq ID No. 263: Amino acid sequence for Wickerhamomyces ciferrii         glucosidase, Uniparc reference UPI000283EB8D, Uniprot reference         K0KVJ2.     -   Seq ID No. 264: Amino acid sequence for Gloeocapsa sp.         glucosidase, Uniparc reference UPI0002A5D085, Uniprot reference         K9XKL8.     -   Seq ID No. 265: Amino acid sequence for Sphaerobacter         thermophilus glucosidase, Uniparc reference UPI0001A3BCB6,         Uniprot reference D1C7U8.     -   Seq ID No. 266: Amino acid sequence for Bacteroides         xylanisolvens glucosidase, Uniparc reference UPI0001CCEF1F,         Uniprot reference D6D4V2.     -   Seq ID No. 267: Amino acid sequence for Prevotella buccae         glucosidase, Uniparc reference UPI0001F149E8, Uniprot reference         E6K4W5.     -   Seq ID No. 268: Amino acid sequence for Grosmannia clavigera         glucosidase, Uniparc reference UPI0001FF1101, Uniprot reference         F0XBR0.     -   Seq ID No. 269: Amino acid sequence for Melampsora         larici-populina glucosidase, Uniparc reference UPI00020F9774,         Uniprot reference F4R4W2.     -   Seq ID No. 270: Amino acid sequence for Prevotella         multisaccharivorax glucosidase, Uniparc reference UPI0002138E9C,         Uniprot reference F8N7G1.     -   Seq ID No. 271: Amino acid sequence for Streptomyces         zinciresistens glucosidase, Uniparc reference UPI0002255A63,         Uniprot reference G2G8K4.     -   Seq ID No. 272: Amino acid sequence for Granulicella mallensis         glucosidase, Uniparc reference UPI0001D9FC40, Uniprot reference         G8NY42.     -   Seq ID No. 273: Amino acid sequence for Gibberella zeae         glucosidase, Uniparc reference UPI00021F1FEE, Uniprot reference         I1RH94.     -   Seq ID No. 274: Amino acid sequence for Modestobacter marinus         glucosidase, Uniparc reference UPI00026090A2, Uniprot reference         I4EYK6.     -   Seq ID No. 275: Amino acid sequence for Lactobacillus         equicursoris glucosidase, Uniparc reference UPI0002869F55,         Uniprot reference K0NRS8.     -   Seq ID No. 276: Amino acid sequence for Colletotrichum         fructicola glucosidase, Uniparc reference UPI0002A93280, Uniprot         reference L2F9W0.     -   Seq ID No. 277: Amino acid sequence for Streptosporangium roseum         glucosidase, Uniparc reference UPI0001BF8AF6, Uniprot reference         D2B261.     -   Seq ID No. 278: Amino acid sequence for Listeria grayi         glucosidase, Uniparc reference UPI00019F252A, Uniprot reference         D7UX19.     -   Seq ID No. 279: Amino acid sequence for Enterococcus italicus         glucosidase, Uniparc reference UPI0001F11EFF, Uniprot reference         E6LF07.     -   Seq ID No. 280: Amino acid sequence for Fluviicola taffensis         glucosidase, Uniparc reference UPI000203D9EA, Uniprot reference         F2IIT6.     -   Seq ID No. 281: Amino acid sequence for Shigella flexneri         glucosidase, Uniparc reference UPI00020CA641, Uniprot reference         F5N4W9.     -   Seq ID No. 282: Amino acid sequence for Actinomyces sp.         glucosidase, Uniparc reference UPI0002189080, Uniprot reference         F9EFW5.     -   Seq ID No. 283: Amino acid sequence for Verticillium dahliae         glucosidase, Uniparc reference UPI00022EBE5A, Uniprot reference         G2X5V6.     -   Seq ID No. 284: Amino acid sequence for Actinoplanes sp.         glucosidase, Uniparc reference UPI00023ED5A8, Uniprot reference         G8S0M0.     -   Seq ID No. 285: Amino acid sequence for Gibberella zeae         glucosidase, Uniparc reference UPI000023EA29, Uniprot reference         I1S320.     -   Seq ID No. 286: Amino acid sequence for Auricularia subglabra         glucosidase, Uniparc reference UPI00027CE685, Uniprot reference         J0WV06.     -   Seq ID No. 287: Amino acid sequence for Nitratireductor indicus         glucosidase, Uniparc reference UPI00028E86D6, Uniprot reference         K2N268.     -   Seq ID No. 288: Amino acid sequence for Thermoclostridium         stercorarium glucosidase, Uniparc reference UPI0002AD999E,         Uniprot reference L7VH66.     -   Seq ID No. 289: Amino acid sequence for Geobacillus sp.         glucosidase, Uniparc reference UPI0002AF2DE3, Uniprot reference         L8A1S2.     -   Seq ID No. 290: Amino acid sequence for uncultured bacterium         glucosidase, Uniparc reference UPI000327C3CC, Uniprot reference         M9ZC55.     -   Seq ID No. 291: Amino acid sequence for Burkholderia ambifaria         glucosidase, Uniparc reference UPI000059ACE1, Uniprot reference         Q0BAK2.     -   Seq ID No. 292: Amino acid sequence for Aspergillus oryzae         glucosidase, Uniparc reference UPI0000676B8D, Uniprot reference         Q2UIR4.     -   Seq ID No. 293: Amino acid sequence for Yersinia         pseudotuberculosis glucosidase, Uniparc reference UPI00004269B5,         Uniprot reference Q66DJ0.     -   Seq ID No. 294: Amino acid sequence for Wallemia ichthyophaga         glucosidase, Uniparc reference UPI000331CC98, Uniprot reference         R9AF64.     -   Seq ID No. 295: Amino acid sequence for Glarea lozoyensis         glucosidase, Uniparc reference UPI0003522404, Uniprot reference         S3DIM6.     -   Seq ID No. 296: Amino acid sequence for Moniliophthora roreri         glucosidase, Uniparc reference UPI0003BF753A, Uniprot reference         V2YU31.     -   Seq ID No. 297: Amino acid sequence for Zhouia amylolytica         glucosidase, Uniparc reference UPI0003DBCB4F, Uniprot reference         W2ULL3.     -   Seq ID No. 298: Amino acid sequence for Fusarium oxysporum         glucosidase, Uniparc reference UPI0003F362FF, Uniprot reference         W9NQQ3.     -   Seq ID No. 299: Amino acid sequence for Flavobacterium         johnsoniae glucosidase, Uniparc reference UPI00006EA168, Uniprot         reference A5FAA5.     -   Seq ID No. 300: Amino acid sequence for Oryza sativa         glucosidase, Uniparc reference UPI00002394F5, Uniprot reference         Q75I93.     -   Seq ID No. 301: Amino acid sequence for Clostridium         saccharoperbutylacetonicum glucosidase, Uniparc reference         UPI0002B65681, Uniprot reference M1MCD1.     -   Seq ID No. 302: Amino acid sequence for Rhodococcus sp.         glucosidase, Uniparc reference UPI0002D21DB1, Uniprot reference         N1MBN6.     -   Seq ID No. 303: Amino acid sequence for Burkholderia ambifaria         glucosidase, Uniparc reference UPI000059C0CD, Uniprot reference         Q0BCV8.     -   Seq ID No. 304: Amino acid sequence for Xanthomonas campestris         glucosidase, Uniparc reference UPI00005CE9E7, Uniprot reference         Q3BVH7.     -   Seq ID No. 305: Amino acid sequence for Caulobacter vibrioides         glucosidase, Uniparc reference UPI00000C7604, Uniprot reference         Q9A6F8.     -   Seq ID No. 306: Amino acid sequence for Arcticibacter         svalbardensis glucosidase, Uniparc reference UPI000338772A,         Uniprot reference R9GRA7.     -   Seq ID No. 307: Amino acid sequence for Winogradskyella         psychrotolerans glucosidase, Uniparc reference UPI00035A4604,         Uniprot reference S7VQ28.     -   Seq ID No. 308: Amino acid sequence for Methyloglobulus morosus         glucosidase, Uniparc reference UPI0003C4ED6A, Uniprot reference         V5DXT8.     -   Seq ID No. 309: Amino acid sequence for Pestalotiopsis fici         glucosidase, Uniparc reference UPI0003E05C04, Uniprot reference         W3WV37.     -   Seq ID No. 310: Amino acid sequence for Capronia coronata         glucosidase, Uniparc reference UPI000434E698, Uniprot reference         W9YNR7.     -   Seq ID No. 311: Amino acid sequence for Aspergillus aculeatus         glucosidase, Uniparc reference UPI00001268FD, Uniprot reference         P48825.     -   Seq ID No. 312: Amino acid sequence for Thermotoga neapolitana         glucosidase, Uniparc reference UPI0000DD5996, Uniprot reference         Q0GC07.     -   Seq ID No. 313: Amino acid sequence for Clostridium         saccharoperbutylacetonicum glucosidase, Uniparc reference         UPI0002B666E8, Uniprot reference M1MJF4.     -   Seq ID No. 314: Amino acid sequence for Schizosaccharomyces         pombe glucosidase, Uniparc reference UPI000006B001, Uniprot         reference O74799.     -   Seq ID No. 315: Amino acid sequence for Phaeosphaeria nodorum         glucosidase, Uniparc reference UPI000161BD2B, Uniprot reference         Q0TXF6.     -   Seq ID No. 316: Amino acid sequence for Xylella fastidiosa Dixon         glucosidase, Uniparc reference UPI00003806C8, Uniprot reference         Q3RGJ3.     -   Seq ID No. 317: Amino acid sequence for Schizosaccharomyces         pombe glucosidase, Uniparc reference UPI000006A330, Uniprot         reference Q9P6J6.     -   Seq ID No. 318: Amino acid sequence for Arcticibacter         svalbardensis glucosidase, Uniparc reference UPI000337E887,         Uniprot reference R9GWD6.     -   Seq ID No. 319: Amino acid sequence for Colletotrichum         gloeosporioides glucosidase, Uniparc reference UPI0003885717,         Uniprot reference T0KJI7.     -   Seq ID No. 320: Amino acid sequence for uncultured bacterium         glucosidase, Uniparc reference UPI0003C9E340, Uniprot reference         V5R1E8.     -   Seq ID No. 321: Amino acid sequence for Xanthomonas arboricola         glucosidase, Uniparc reference UPI0003E06A61, Uniprot reference         W4S7I5.     -   Seq ID No. 322: Amino acid sequence for Fusarium oxysporum         glucosidase, Uniparc reference UPI00021EC697, Uniprot reference         X0A8X8.     -   Seq ID No. 323: Amino acid sequence for Paenibacillus polymyxa         glucosidase, Uniparc reference UPI00001108D0, Uniprot reference         P22073.     -   Seq ID No. 324: Amino acid sequence for Kluyveromyces marxianus         glucosidase, Uniparc reference UPI0001BE5ADA, Uniprot reference         D1GCC6.     -   Seq ID No. 325: Amino acid sequence for Ilumatobacter coccineus         glucosidase, Uniparc reference UPI0002C04A25, Uniprot reference         M5A594.     -   Seq ID No. 326: Amino acid sequence for Agrobacterium sp.         glucosidase, Uniparc reference UPI0000126912, Uniprot reference         P12614.     -   Seq ID No. 327: Amino acid sequence for Cytophaga hutchinsonii         glucosidase, Uniparc reference UPI000038ECF7, Uniprot reference         Q11P53.     -   Seq ID No. 328: Amino acid sequence for Thermobifida fusca         glucosidase, Uniparc reference UPI00003C5CA4, Uniprot reference         Q47PF5.     -   Seq ID No. 329: Amino acid sequence for Botryotinia fuckeliana         glucosidase, Uniparc reference UPI0000069E5E, Uniprot reference         Q9UVJ6.     -   Seq ID No. 330: Amino acid sequence for Agarivorans albus         glucosidase, Uniparc reference UPI00033994D2, Uniprot reference         R9PTK2.     -   Seq ID No. 331: Amino acid sequence for Enterococcus sp.         glucosidase, Uniparc reference UPI00038B7639, Uniprot reference         T0UDU2.     -   Seq ID No. 332: Amino acid sequence for Salinispira pacifica         glucosidase, Uniparc reference UPI0003D8A7DC, Uniprot reference         V5WKT4.     -   Seq ID No. 333: Amino acid sequence for Bacteroides         xylanisolvens glucosidase, Uniparc reference UPI0003ECF15E,         Uniprot reference W6P696.     -   Seq ID No. 334: Amino acid sequence for Fusarium oxysporum         glucosidase, Uniparc reference UPI000430029A, Uniprot reference         X0AB46.     -   Seq ID No. 335: Amino acid sequence for Thermotoga maritima         glucosidase, Uniparc reference UPI0000126906, Uniprot reference         Q08638B.     -   Seq ID No. 336: Amino acid sequence for Neotermes koshunensis         glucosidase, Uniparc reference UPI0000083EA8, Uniprot reference         Q8T0W7.     -   Seq ID No. 337: Amino acid sequence for Thanatephorus cucumeris         glucosidase, Uniparc reference UPI0002BF34B3, Uniprot reference         M5CHG9.     -   Seq ID No. 338: Amino acid sequence for Hungateiclostridium         thermocellum glucosidase, Uniparc reference UPI000053581D,         Uniprot reference P14002.     -   Seq ID No. 339: Amino acid sequence for Koribacter versatilis         glucosidase, Uniparc reference UPI0000D76A70, Uniprot reference         Q1IJ89.     -   Seq ID No. 340: Amino acid sequence for Neosartorya fumigata         glucosidase, Uniparc reference UPI00005203D8, Uniprot reference         Q4WLX5.     -   Seq ID No. 341: Amino acid sequence for Amycolatopsis         vancoresmycina glucosidase, Uniparc reference UPI00032DB8C1,         Uniprot reference R1FKF0.     -   Seq ID No. 342: Amino acid sequence for Gibberella fujikuroi         glucosidase, Uniparc reference UPI0003519C76, Uniprot reference         S0EKU1.     -   Seq ID No. 343: Amino acid sequence for Bifidobacterium longum         glucosidase, Uniparc reference UPI000390F839, Uniprot reference         T2I2H5.     -   Seq ID No. 344: Amino acid sequence for uncultured bacterium         glucosidase, Uniparc reference UPI0003DFF0DA, Uniprot reference         W0FLD1.     -   Seq ID No. 345: Amino acid sequence for Bacteroides         xylanisolvens glucosidase, Uniparc reference UPI0002D3D994,         Uniprot reference W6P9J9.     -   Seq ID No. 346: Amino acid sequence for Fusarium oxysporum         glucosidase, Uniparc reference UPI00042F8016, Uniprot reference         X0HA35.     -   Seq ID No. 347: Amino acid sequence for Sorghum bicolor         glucosidase, Uniparc reference UPI00000A7F40, Uniprot reference         Q41290.     -   Seq ID No. 348: Amino acid sequence for Clostridium         cellulovorans glucosidase, Uniparc reference UPI000050B701,         Uniprot reference Q53EH2.     -   Seq ID No. 349: Amino acid sequence for Dacryopinax primogenitus         glucosidase, Uniparc reference UPI0002C29EAA, Uniprot reference         M5G1U5.     -   Seq ID No. 350: Amino acid sequence for Rhizobium radiobacter         glucosidase, Uniparc reference UPI0000126913, Uniprot reference         P27034.     -   Seq ID No. 351: Amino acid sequence for Phanerochaete         chrysosporium glucosidase, Uniparc reference UPI00006E0CC7,         Uniprot reference Q25BW5A.     -   Seq ID No. 352: Amino acid sequence for Enterobacter agglomerans         glucosidase, Uniparc reference UPI0000126905, Uniprot reference         Q59437.     -   Seq ID No. 353: Amino acid sequence for Candidatus microthrix         glucosidase, Uniparc reference UPI00033056C4, Uniprot reference         R4YWU4.     -   Seq ID No. 354: Amino acid sequence for Chthonomonas calidirosea         glucosidase, Uniparc reference UPI0003427F29, Uniprot reference         S0ET37.     -   Seq ID No. 355: Amino acid sequence for Acholeplasma brassicae         glucosidase, Uniparc reference UPI0003B04208, Uniprot reference         U4KMR7.     -   Seq ID No. 356: Amino acid sequence for Chania         multitudinisentens glucosidase, Uniparc reference UPI0003E13BD3,         Uniprot reference W0LJ28.     -   Seq ID No. 357: Amino acid sequence for Gibberella moniliformis         glucosidase, Uniparc reference UPI0003ECCF80, Uniprot reference         W7MW98.     -   Seq ID No. 358: Amino acid sequence for Fusarium oxysporum         glucosidase, Uniparc reference UPI0004305856, Uniprot reference         X0I2U8.     -   Seq ID No. 359: Amino acid sequence for Phanerochaete         chrysosporium glucosidase, Uniparc reference UPI00006E0CC7,         Uniprot reference Q25BW5B.     -   Seq ID No. 360: Amino acid sequence for Secale cereale         glucosidase, Uniparc reference UPI00000A7EFC, Uniprot reference         Q9FYS3.     -   Seq ID No. 361: Amino acid sequence for Anoxybacillus gonensis         glucosidase, Uniparc reference UPI0002C0273E, Uniprot reference         M5QUM2.     -   Seq ID No. 362: Amino acid sequence for Thermotoga maritima         glucosidase, Uniparc reference UPI0000126906, Uniprot reference         Q08638A.     -   Seq ID No. 363: Amino acid sequence for Rhodospirillum rubrum         glucosidase, Uniparc reference UPI00003C2ACC, Uniprot reference         Q2RP51.     -   Seq ID No. 364: Amino acid sequence for Thermotoga neapolitana         glucosidase, Uniparc reference UPI00000B9013, Uniprot reference         Q60038.     -   Seq ID No. 365: Amino acid sequence for Candidatus microthrix         glucosidase, Uniparc reference UPI00032F466E, Uniprot reference         R4Z6M6.     -   Seq ID No. 366: Amino acid sequence for Ruminiclostridium         cellobioparum glucosidase, Uniparc reference UPI00032874EC,         Uniprot reference S0FPI8.     -   Seq ID No. 367: Amino acid sequence for Acholeplasma brassicae         glucosidase, Uniparc reference UPI0003B0490C, Uniprot reference         U4KMV4.     -   Seq ID No. 368: Amino acid sequence for Klebsiella pneumoniae         glucosidase, Uniparc reference UPI0003DB6EFD, Uniprot reference         W1BBP5.     -   Seq ID No. 369: Amino acid sequence for Enterobacter sp.         glucosidase, Uniparc reference UPI0003ED0A8E, Uniprot reference         W7P6Y1.     -   Seq ID No. 370: Amino acid sequence for Rhizoctonia solani         glucosidase, Uniparc reference UPI00045BB507, Uniprot reference         X8JI88.     -   Seq ID No. 371: Amino acid sequence for Homo sapiens         glucosidase, Uniparc reference UPI0000072C73, Uniprot reference         Q9H227.     -   Seq ID No. 372: Amino acid sequence for uncultured bacterium         glucosidase, Uniparc reference UPI0000DD5304, Uniprot reference         Q0GMU3.     -   Seq ID No. 373: Amino acid sequence for Thermoanaerobacter         thermohydrosulfuricus glucosidase, Uniparc reference         UPI0002CA9E4F, Uniprot reference M8CQD9.     -   Seq ID No. 374: Amino acid sequence for Stigmatella aurantiaca         glucosidase, Uniparc reference UPI0000E297D8, Uniprot reference         Q091M8.     -   Seq ID No. 375: Amino acid sequence for Burkholderia         thailandensis glucosidase, Uniparc reference UPI00006676B1,         Uniprot reference Q2T7C9.     -   Seq ID No. 376: Amino acid sequence for Yersinia         pseudotuberculosis glucosidase, Uniparc reference UPI0000426E8F,         Uniprot reference Q665S2.     -   Seq ID No. 377: Amino acid sequence for Togninia minima         glucosidase, Uniparc reference UPI00032BF71F, Uniprot reference         R8BQM8.     -   Seq ID No. 378: Amino acid sequence for Ophiostoma piceae         glucosidase, Uniparc reference UPI00035213C2, Uniprot reference         S3BVJ8.     -   Seq ID No. 379: Amino acid sequence for Pyronema omphalodes         glucosidase, Uniparc reference UPI0003B117B7, Uniprot reference         U4LIL3.     -   Seq ID No. 380: Amino acid sequence for Ogataea parapolymorpha         glucosidase, Uniparc reference UPI0001F76D1F, Uniprot reference         W1QIW0.     -   Seq ID No. 381: Amino acid sequence for Hymenobacter swuensis         glucosidase, Uniparc reference UPI0003F2059F, Uniprot reference         W8F6U0.     -   Seq ID No. 382: Amino acid sequence for Flavobacterium         johnsoniae glucosidase, Uniparc reference UPI00006E8E1B, Uniprot         reference A5FEF5.     -   Seq ID No. 383: Amino acid sequence for Paenibacillus polymyxa         glucosidase, Uniparc reference UPI000012690B, Uniprot reference         P22505.     -   Seq ID No. 384: Amino acid sequence for Oryza sativa         glucosidase, Uniparc reference UPI000009D014, Uniprot reference         Q8L7J2.     -   Seq ID No. 385: Amino acid sequence for Oryza sativa         glucosidase, Uniparc reference UPI0000E580F2, Uniprot reference         B8AVF0.     -   Seq ID No. 386: Amino acid sequence for Nannochloris         glucosidase, Uniparc reference UPI000EA7F16F, Uniprot reference         A0A452CSM4.     -   Seq ID No. 387: Amino acid sequence for Halothermothrix orenii         glucosidase, Uniparc reference UPI00006AE508, Uniprot reference         B8CYA8.     -   Seq ID No. 388: Amino acid sequence for Neurospora crassa         glucosidase, Uniparc reference UPI000018B2B4, Uniprot reference         Q7RWP2.     -   Seq ID No. 389: Amino acid sequence for Micrococcus antarcticus         glucosidase, Uniparc reference UPI000192BB5F, Uniprot reference         B9V8P5.     -   Seq ID No. 390: Amino acid sequence for Exiguobacterium         antarcticum glucosidase, Uniparc reference UPI000285E79E,         Uniprot reference K0A8J9.     -   Seq ID No. 391: Amino acid sequence for Thermus thermophilus         glucosidase, Uniparc reference UPI00000BEB61, Uniprot reference         Q9RA61.     -   Seq ID No. 392: Amino acid sequence for Trichoderma harzianum         glucosidase, Uniparc reference UPI00078BF747, Uniprot reference         A0A2T4AR08.     -   Seq ID No. 393: Amino acid sequence for Hypocrea jecorina         glucosidase, Uniparc reference UPI000006AA61, Uniprot reference         Q12715.     -   Seq ID No. 394: Amino acid sequence for Streptomyces sp.         glucosidase, Uniparc reference UPI00000B411B, Uniprot reference         Q59976.     -   Seq ID No. 395: Amino acid sequence for Streptococcus pyogenes         glucosidase, Uniparc reference UPI00000C7E56, Uniprot reference         Q99YP9.     -   Seq ID No. 396: Amino acid sequence for Trifolium repens         glucosidase, Uniparc reference UPI000012691B, Uniprot reference         P26205.     -   Seq ID No. 397: Amino acid sequence for Talaromyces emersonii         glucosidase, Uniparc reference UPI000006C8FF, Uniprot reference         Q8TGI8.     -   Seq ID No. 398: Amino acid sequence for Hungateiclostridium         thermocellum glucosidase, Uniparc reference UPI0000126903,         Uniprot reference P26208.     -   Seq ID No. 399: Amino acid sequence for Lactobacillus plantarum         glucosidase, Uniparc reference UPI000219FE3E, Uniprot reference         F9ULH8.     -   Seq ID No. 400: Amino acid sequence for Agrobacterium         tumefaciens glucosidase, Uniparc reference UPI0003F2033A,         Uniprot reference A0A2I4PGZ0.     -   Seq ID No. 401: Codon optimised DNA encoding 6×His Cyberlindnera         fabianii glucosidase, Uniparc reference UPI00049B1A8C, Uniprot         reference A0A061B3J2.     -   Seq ID No. 402: Codon optimised DNA encoding 6×His         Flavobacterium gilvum glucosidase, Uniparc reference         UPI0004E3EF7B, Uniprot reference A0A085EII0.     -   Seq ID No. 403: Codon optimised DNA encoding 6×His Algibacter         lectus glucosidase, Uniparc reference UPI00050EE490, Uniprot         reference A0A090X649.     -   Seq ID No. 404: Codon optimised DNA encoding 6×His         Microbacterium azadirachtae glucosidase, Uniparc reference         UPI0005ECB51E, Uniprot reference A0A0F0LB94.     -   Seq ID No. 405: Codon optimised DNA encoding 6×His         Actinobacteria bacterium glucosidase, Uniparc reference         UPI0006588DAD, Uniprot reference A0A0J0UT37.     -   Seq ID No. 406: Codon optimised DNA encoding 6×His Chloroflexi         bacterium glucosidase, Uniparc reference UPI0007968552, Uniprot         reference A0A136KWB3.     -   Seq ID No. 407: Codon optimised DNA encoding 6×His         Komagataeibacter rhaeticus glucosidase, Uniparc reference         UPI0002080410, Uniprot reference A0A181C809.     -   Seq ID No. 408: Codon optimised DNA encoding 6×His Bacteroides         sp. glucosidase, Uniparc reference UPI0008211BFC, Uniprot         reference A0A1C5WEL8.     -   Seq ID No. 409: Codon optimised DNA encoding 6×His Streptomyces         rubrolavendulae glucosidase, Uniparc reference UPI00085A2BD0,         Uniprot reference A0A1D8FZW3.     -   Seq ID No. 410: Codon optimised DNA encoding 6×His Clostridium         roseum glucosidase, Uniparc reference UPI00098C60F6, Uniprot         reference A0A1S8KYM5.     -   Seq ID No. 411: Codon optimised DNA encoding 6×His uncultured         bacterium glucosidase, Uniparc reference UPI0009CE0D4C, Uniprot         reference A0A1V5M6V6.     -   Seq ID No. 412: Codon optimised DNA encoding 6×His Firmicutes         bacterium glucosidase, Uniparc reference UPI0009D4127D, Uniprot         reference A0A1V6AN95.     -   Seq ID No. 413: Codon optimised DNA encoding 6×His         Anthracocystis flocculosa glucosidase, Uniparc reference         UPI00045601AB, Uniprot reference A0A061H1Z3.     -   Seq ID No. 414: Codon optimised DNA encoding 6×His         Bifidobacterium boum glucosidase, Uniparc reference         UPI0004FF77C7, Uniprot reference A0A086ZKU2.     -   Seq ID No. 415: Codon optimised DNA encoding 6×His Jejuia         pallidilutea glucosidase, Uniparc reference UPI00051EDBDE,         Uniprot reference A0A098LTR2.     -   Seq ID No. 416: Codon optimised DNA encoding 6×His Ceratocystis         fimbriata glucosidase, Uniparc reference UPI00062105AB, Uniprot         reference A0A0F8B2B0.     -   Seq ID No. 417: Codon optimised DNA encoding 6×His         Actinobacteria bacterium glucosidase, Uniparc reference         UPI0006583AB1, Uniprot reference A0A0J0UVW7.     -   Seq ID No. 418: Codon optimised DNA encoding 6×His Rhodococcus         sp. glucosidase, Uniparc reference UPI0007AABFAD, Uniprot         reference A0A143QAX3.     -   Seq ID No. 419: Codon optimised DNA encoding 6×His Valsa mali         glucosidase, Uniparc reference UPI0007F2D02D, Uniprot reference         A0A194VF47.     -   Seq ID No. 420: Codon optimised DNA encoding 6×His uncultured         Bacteroides sp. glucosidase, Uniparc reference UPI000821004D,         Uniprot reference A0A1C5WSI4.     -   Seq ID No. 421: Codon optimised DNA encoding 6×His         Eisenbergiella tayi glucosidase, Uniparc reference         UPI00084089B2, Uniprot reference A0A1E3ALT2.     -   Seq ID No. 422: Codon optimised DNA encoding 6×His Streptomyces         sp. glucosidase, Uniparc reference UPI000978E914, Uniprot         reference A0A1V2MY14.     -   Seq ID No. 423: Codon optimised DNA encoding 6×His Firmicutes         bacterium glucosidase, Uniparc reference UPI0009CBF21C, Uniprot         reference A0A1V5MH90.     -   Seq ID No. 424: Codon optimised DNA encoding 6×His Tenericutes         bacterium glucosidase, Uniparc reference UPI0009D5B1F0, Uniprot         reference A0A1V6BAK3.     -   Seq ID No. 425: Codon optimised DNA encoding 6×His Gluconobacter         oxydans glucosidase, Uniparc reference UPI0004A87350, Uniprot         reference A0A067Z479.     -   Seq ID No. 426: Codon optimised DNA encoding 6×His         Bifidobacterium catenulatum glucosidase, Uniparc reference         UPI00050787A2, Uniprot reference A0A087B8Q8.     -   Seq ID No. 427: Codon optimised DNA encoding 6×His Bionectria         ochroleuca glucosidase, Uniparc reference UPI0005965863, Uniprot         reference A0A0B7K538.     -   Seq ID No. 428: Codon optimised DNA encoding 6×His Parcubacteria         sp. glucosidase, Uniparc reference UPI0006377CA4, Uniprot         reference A0A0G0GD78.     -   Seq ID No. 429: Codon optimised DNA encoding 6×His         Microbacterium ketosireducens glucosidase, Uniparc reference         UPI0006228575, Uniprot reference A0A0M2H276.     -   Seq ID No. 430: Codon optimised DNA encoding 6×His Roseburia         faecis glucosidase, Uniparc reference UPI0006C454B4, Uniprot         reference A0A173R3W4.     -   Seq ID No. 431: Codon optimised DNA encoding 6×His Kwoniella         dejecticola glucosidase, Uniparc reference UPI0007F1D695,         Uniprot reference A0A1A6A050.     -   Seq ID No. 432: Codon optimised DNA encoding 6×His uncultured         Clostridium sp. glucosidase, Uniparc reference UPI0008232A70,         Uniprot reference A0A1C6C862.     -   Seq ID No. 433: Codon optimised DNA encoding 6×His Cyberlindnera         jadinii glucosidase, Uniparc reference UPI000866CB30, Uniprot         reference A0A1E4S2F8.     -   Seq ID No. 434: Codon optimised DNA encoding 6×His Bacteroidetes         bacterium glucosidase, Uniparc reference UPI0009D3483D, Uniprot         reference A0A1V5G4W6.     -   Seq ID No. 435: Codon optimised DNA encoding 6×His         Verrucomicrobia bacterium glucosidase, Uniparc reference         UPI0009CC9AEA, Uniprot reference A0A1V5Q4R4.     -   Seq ID No. 436: Codon optimised DNA encoding 6×His Bacteroidetes         bacterium glucosidase, Uniparc reference UPI0009C6DE73, Uniprot         reference A0A1V6BV25.     -   Seq ID No. 437: Codon optimised DNA encoding 6×His Lichtheimia         ramosa glucosidase, Uniparc reference UPI0004E051A9, Uniprot         reference A0A077WUK7.     -   Seq ID No. 438: Codon optimised DNA encoding 6×His         Bifidobacterium mongoliense glucosidase, Uniparc reference         UPI0005060F52, Uniprot reference A0A087BWT7.     -   Seq ID No. 439: Codon optimised DNA encoding 6×His Vibrio         ishigakensis glucosidase, Uniparc reference UPI0005910ED9,         Uniprot reference A0A0B8NZY1.     -   Seq ID No. 440: Codon optimised DNA encoding 6×His Phaeomoniella         chlamydospora glucosidase, Uniparc reference UPI00063B706F,         Uniprot reference A0A0G2HEV5.     -   Seq ID No. 441: Codon optimised DNA encoding 6×His Ardenticatena         maritima glucosidase, Uniparc reference UPI0006C04F59, Uniprot         reference A0A0M8K5H7.     -   Seq ID No. 442: Codon optimised DNA encoding 6×His Coprococcus         comes glucosidase, Uniparc reference UPI000197E031, Uniprot         reference A0A173WPC4.     -   Seq ID No. 443: Codon optimised DNA encoding 6×His Nocardioides         dokdonensis glucosidase, Uniparc reference UPI0007DDBAB3,         Uniprot reference A0A1A9GNJ0.     -   Seq ID No. 444: Codon optimised DNA encoding 6×His uncultured         Clostridium sp. glucosidase, Uniparc reference UPI000822F7EB,         Uniprot reference A0A1C6EBC1.     -   Seq ID No. 445: Codon optimised DNA encoding 6×His         Acetobacterium wieringae glucosidase, Uniparc reference         UPI000878EB40, Uniprot reference A0A1F2PFB4.     -   Seq ID No. 446: Codon optimised DNA encoding 6×His Tenericutes         bacterium glucosidase, Uniparc reference UPI0009D0A2C1, Uniprot         reference A0A1V5HNJ3.     -   Seq ID No. 447: Codon optimised DNA encoding 6×His Tenericutes         bacterium glucosidase, Uniparc reference UPI0009CB8A73, Uniprot         reference A0A1V5UFI1.     -   Seq ID No. 448: Codon optimised DNA encoding 6×His uncultured         bacterium glucosidase, Uniparc reference UPI0009CC90AF, Uniprot         reference A0A1V6CDT2.     -   Seq ID No. 449: Codon optimised DNA encoding 6×His         Parabacteroides distasonis glucosidase, Uniparc reference         UPI0004D8E473, Uniprot reference A0A078SYD0.     -   Seq ID No. 450: Codon optimised DNA encoding 6×His         Bifidobacterium psychraerophilum glucosidase, Uniparc reference         UPI0005006C6A, Uniprot reference A0A087CJB0.     -   Seq ID No. 451: Codon optimised DNA encoding 6×His Hebeloma         cylindrosporum glucosidase, Uniparc reference UPI00059A3BA8,         Uniprot reference A0A0C2YJF2.     -   Seq ID No. 452: Codon optimised DNA encoding 6×His Brenneria         goodwinii glucosidase, Uniparc reference UPI0006579FEA, Uniprot         reference A0A0G4JRR8.     -   Seq ID No. 453: Codon optimised DNA encoding 6×His Aspergillus         calidoustus glucosidase, Uniparc reference UPI00073C9E60,         Uniprot reference A0A0U5GN20.     -   Seq ID No. 454: Codon optimised DNA encoding 6×His Bacteroides         finegoldii glucosidase, Uniparc reference UPI0006C6E0C3, Uniprot         reference A0A174BEZ2.     -   Seq ID No. 455: Codon optimised DNA encoding 6×His         Altererythrobacter dongtanensis glucosidase, Uniparc reference         UPI0008153D6D, Uniprot reference A0A1B2A943.     -   Seq ID No. 456: Codon optimised DNA encoding 6×His uncultured         Anaerotruncus sp. glucosidase, Uniparc reference UPI0008206E7B,         Uniprot reference A0A1C6FWD9.     -   Seq ID No. 457: Codon optimised DNA encoding 6×His Candidatus         firestone glucosidase, Uniparc reference UPI0008AC0E17, Uniprot         reference A0A1F5UJR6.     -   Seq ID No. 458: Codon optimised DNA encoding 6×His Tenericutes         bacterium glucosidase, Uniparc reference UPI0009C6C8E4, Uniprot         reference A0A1V5HS22.     -   Seq ID No. 459: Codon optimised DNA encoding 6×His Lentisphaerae         bacterium glucosidase, Uniparc reference UPI0009CEE1AB, Uniprot         reference A0A1V5VBL2.     -   Seq ID No. 460: Codon optimised DNA encoding 6×His         Planctomycetes bacterium glucosidase, Uniparc reference         UPI0009CAAB0A, Uniprot reference A0A1V6FZ47.     -   Seq ID No. 461: Codon optimised DNA encoding 6×His         Pseudallescheria apiosperma glucosidase, Uniparc reference         UPI0004DD62AC, Uniprot reference A0A084G332.     -   Seq ID No. 462: Codon optimised DNA encoding 6×His Nonlabens         sediminis glucosidase, Uniparc reference UPI000507F00A, Uniprot         reference A0A090Q4N8.     -   Seq ID No. 463: Codon optimised DNA encoding 6×His Gynuella         sunshinyii glucosidase, Uniparc reference UPI0005CC42CA, Uniprot         reference A0A0C5VDU3.     -   Seq ID No. 464: Codon optimised DNA encoding 6×His Verticillium         longisporum glucosidase, Uniparc reference UPI00063E4005,         Uniprot reference A0A0G4N9Q7.     -   Seq ID No. 465: Codon optimised DNA encoding 6×His Cellulomonas         sp. glucosidase, Uniparc reference UPI00073C6CD3, Uniprot         reference A0A0V8TAB5.     -   Seq ID No. 466: Codon optimised DNA encoding 6×His Hungatella         hathewayi glucosidase, Uniparc reference UPI0006C024BB, Uniprot         reference A0A174FBX7.     -   Seq ID No. 467: Codon optimised DNA encoding 6×His Mesorhizobium         sp. glucosidase, Uniparc reference UPI000688E74D, Uniprot         reference A0A1C2DG64.     -   Seq ID No. 468: Codon optimised DNA encoding 6×His Clostridium         sp. glucosidase, Uniparc reference UPI000822FAB7, Uniprot         reference A0A1C6GRT5.     -   Seq ID No. 469: Codon optimised DNA encoding 6×His Chlamydiales         bacterium glucosidase, Uniparc reference UPI0009284E74, Uniprot         reference A0A1M3CSY6.     -   Seq ID No. 470: Codon optimised DNA encoding 6×His Spirochaetes         bacterium glucosidase, Uniparc reference UPI0009CB461D, Uniprot         reference A0A1V5HUX3.     -   Seq ID No. 471: Codon optimised DNA encoding 6×His bacterium         glucosidase, Uniparc reference UPI0009C92D6A, Uniprot reference         A0A1V5VHB9.     -   Seq ID No. 472: Codon optimised DNA encoding 6×His Thermotogae         bacterium glucosidase, Uniparc reference UPI0009D12B86, Uniprot         reference A0A1V6H2W5.     -   Seq ID No. 473: Codon optimised DNA encoding 6×His         Pseudallescheria apiosperma glucosidase, Uniparc reference         UPI0004DCBED3, Uniprot reference A0A084GGE2.     -   Seq ID No. 474: Codon optimised DNA encoding 6×His Algibacter         lectus glucosidase, Uniparc reference UPI00050E0BE3, Uniprot         reference A0A090VF17.     -   Seq ID No. 475: Codon optimised DNA encoding 6×His Paxillus         involutus glucosidase, Uniparc reference UPI0005B075C8, Uniprot         reference A0A0C9TWP5.     -   Seq ID No. 476: Codon optimised DNA encoding 6×His Verticillium         longisporum glucosidase, Uniparc reference UPI00063DF006,         Uniprot reference A0A0G4NA55.     -   Seq ID No. 477: Codon optimised DNA encoding 6×His         Mucilaginibacter gotjawali glucosidase, Uniparc reference         UPI00076F8EA4, Uniprot reference A0A110B1H1.     -   Seq ID No. 478: Codon optimised DNA encoding 6×His Bacteroides         uniformis glucosidase, Uniparc reference UPI0006C0FFEF, Uniprot         reference A0A174IWW4.     -   Seq ID No. 479: Codon optimised DNA encoding 6×His Coprococcus         sp. glucosidase, Uniparc reference UPI0008222B77, Uniprot         reference A0A1C5W0N6.     -   Seq ID No. 480: Codon optimised DNA encoding 6×His Blautia sp.         glucosidase, Uniparc reference UPI0006C36823, Uniprot reference         A0A1C6K2X5.     -   Seq ID No. 481: Codon optimised DNA encoding 6×His Cellulomonas         sp. glucosidase, Uniparc reference UPI00092B3CD4, Uniprot         reference A0A1M3ELH4.     -   Seq ID No. 482: Codon optimised DNA encoding 6×His bacterium         glucosidase, Uniparc reference UPI0009C85428, Uniprot reference         A0A1V5J984.     -   Seq ID No. 483: Codon optimised DNA encoding 6×His Spirochaetes         bacterium glucosidase, Uniparc reference UPI0009CA1D5C, Uniprot         reference A0A1V5WHQ7.     -   Seq ID No. 484: Codon optimised DNA encoding 6×His Tenericutes         bacterium glucosidase, Uniparc reference UPI0009C7041F, Uniprot         reference A0A1V6IJ57.     -   Seq ID No. 485: Codon optimised DNA encoding 6×His         Flavobacterium gilvum glucosidase, Uniparc reference         UPI0004E2A41F, Uniprot reference A0A085EG29.     -   Seq ID No. 486: Codon optimised DNA encoding 6×His Algibacter         lectus glucosidase, Uniparc reference UPI0005102470, Uniprot         reference A0A090WWZ1.     -   Seq ID No. 487: Codon optimised DNA encoding 6×His Hydnomerulius         pinastri glucosidase, Uniparc reference UPI0005B0F0AE, Uniprot         reference A0A0C9WDY0.     -   Seq ID No. 488: Codon optimised DNA encoding 6×His Nocardia         farcinica glucosidase, Uniparc reference UPI00065C2666, Uniprot         reference A0A0H5NWN2.     -   Seq ID No. 489: Codon optimised DNA encoding 6×His Bacteroides         cellulosilyticus glucosidase, Uniparc reference UPI000760375B,         Uniprot reference A0A125MG18.     -   Seq ID No. 490: Codon optimised DNA encoding 6×His Fonsecaea         erecta glucosidase, Uniparc reference UPI0007DF4250, Uniprot         reference A0A178ZBN2.     -   Seq ID No. 491: Codon optimised DNA encoding 6×His Bacteroides         sp. glucosidase, Uniparc reference UPI0008209852, Uniprot         reference A0A1C5W9N9.     -   Seq ID No. 492: Codon optimised DNA encoding 6×His Tannerella         forsythia glucosidase, Uniparc reference UPI00086C34C1, Uniprot         reference A0A1D3UGH8.     -   Seq ID No. 493: Codon optimised DNA encoding 6×His         Microbacterium esteraromaticum glucosidase, Uniparc reference         UPI00097E83BB, Uniprot reference A0A1R4KI92.     -   Seq ID No. 494: Codon optimised DNA encoding 6×His bacterium         glucosidase, Uniparc reference UPI0009D255E5, Uniprot reference         A0A1V5LJK9.     -   Seq ID No. 495: Codon optimised DNA encoding 6×His Candidatus         hydrogenedentes glucosidase, Uniparc reference UPI0009C5A3CF,         Uniprot reference A0A1V5Z2L2.     -   Seq ID No. 496: Codon optimised DNA encoding 6×His Bacteroidetes         bacterium glucosidase, Uniparc reference UPI0009C55799, Uniprot         reference A0A1V6J4J8.     -   Seq ID No. 497: Codon optimised DNA encoding 6×His Penicillium         solitum glucosidase, Uniparc reference UPI0009D4067F, Uniprot         reference A0A1V6RQ41.     -   Seq ID No. 498: Codon optimised DNA encoding 6×His Weissella         soli glucosidase, Uniparc reference UPI0008737AA2, Uniprot         reference A0A288Q8I2.     -   Seq ID No. 499: Codon optimised DNA encoding 6×His Acetatifactor         muris glucosidase, Uniparc reference UPI000CAC57D4, Uniprot         reference A0A2K4ZN91.     -   Seq ID No. 500: Codon optimised DNA encoding 6×His Corynespora         cassiicola glucosidase, Uniparc reference UPI000D237A4A, Uniprot         reference A0A2T2NYD4.     -   Seq ID No. 501: Codon optimised DNA encoding 6×His Meira         miltonrushii glucosidase, Uniparc reference UPI000D77C91D,         Uniprot reference A0A316V6M3.     -   Seq ID No. 502: Codon optimised DNA encoding 6×His Bacteroides         fragilis glucosidase, Uniparc reference UPI00004E1F76, Uniprot         reference A0A380YVC7.     -   Seq ID No. 503: Codon optimised DNA encoding 6×His Malassezia         restricta glucosidase, Uniparc reference UPI000F0C30E8, Uniprot         reference A0A3G2SB79.     -   Seq ID No. 504: Codon optimised DNA encoding 6×His Fusarium         euwallaceae glucosidase, Uniparc reference UPI000FFFEFB2,         Uniprot reference A0A430LYA2.     -   Seq ID No. 505: Codon optimised DNA encoding 6×His Psathyrella         aberdarensis glucosidase, Uniparc reference UPI0010251887,         Uniprot reference A0A4Q2E070.     -   Seq ID No. 506: Codon optimised DNA encoding 6×His Aeromonas         hydrophila glucosidase, Uniparc reference UPI0000E69509, Uniprot         reference A0KLP6.     -   Seq ID No. 507: Codon optimised DNA encoding 6×His         Saccharopolyspora erythraea glucosidase, Uniparc reference         UPI00000B86CB, Uniprot reference A4F7P9.     -   Seq ID No. 508: Codon optimised DNA encoding 6×His Streptomyces         sviceus glucosidase, Uniparc reference UPI000180240E, Uniprot         reference B5I181.     -   Seq ID No. 509: Codon optimised DNA encoding 6×His Naematelia         encephala glucosidase, Uniparc reference UPI000A250F78, Uniprot         reference A0A1Y2AWB7.     -   Seq ID No. 510: Codon optimised DNA encoding 6×His         Hartmannibacter diazotrophicus glucosidase, Uniparc reference         UPI000C0221F1, Uniprot reference A0A2C9D6I2.     -   Seq ID No. 511: Codon optimised DNA encoding 6×His Pontimonas         salivibrio glucosidase, Uniparc reference UPI000CEB5AB1, Uniprot         reference A0A2L2BPE2.     -   Seq ID No. 512: Codon optimised DNA encoding 6×His Cadophora sp.         glucosidase, Uniparc reference UPI000D5B9C38, Uniprot reference         A0A2V1CH24.     -   Seq ID No. 513: Codon optimised DNA encoding 6×His Meira         miltonrushii glucosidase, Uniparc reference UPI000D779558,         Uniprot reference A0A316V8S0.     -   Seq ID No. 514: Codon optimised DNA encoding 6×His Monilinia         fructigena glucosidase, Uniparc reference UPI000DC42E3D, Uniprot         reference A0A395IJW4.     -   Seq ID No. 515: Codon optimised DNA encoding 6×His Hortaea         werneckii glucosidase, Uniparc reference UPI000F3E476C, Uniprot         reference A0A3M6XGS0.     -   Seq ID No. 516: Codon optimised DNA encoding 6×His Streptomyces         netropsis glucosidase, Uniparc reference UPI00101460D7, Uniprot         reference A0A445N7U0.     -   Seq ID No. 517: Codon optimised DNA encoding 6×His Aureobasidium         pullulans glucosidase, Uniparc reference UPI001139C6A8, Uniprot         reference A0A4S9IFI0.     -   Seq ID No. 518: Codon optimised DNA encoding 6×His Aspergillus         clavatus glucosidase, Uniparc reference UPI0000EA5CFF, Uniprot         reference A1CTN9.     -   Seq ID No. 519: Codon optimised DNA encoding 6×His Clavibacter         michiganensis glucosidase, Uniparc reference UPI0001523037,         Uniprot reference A5CT94.     -   Seq ID No. 520: Codon optimised DNA encoding 6×His Penicillium         rubens glucosidase, Uniparc reference UPI0001831CF5, Uniprot         reference B6H7R5.     -   Seq ID No. 521: Codon optimised DNA encoding 6×His         Lachnoclostridium sp. glucosidase, Uniparc reference         UPI000B365547, Uniprot reference A0A1Y4NTL9.     -   Seq ID No. 522: Codon optimised DNA encoding 6×His         Rhodobacteraceae bacterium glucosidase, Uniparc reference         UPI000C09BF88, Uniprot reference A0A2D5IXB9.     -   Seq ID No. 523: Codon optimised DNA encoding 6×His Bacteroides         fragilis glucosidase, Uniparc reference UPI0004B5EEF2, Uniprot         reference A0A2M9UUC4.     -   Seq ID No. 524: Codon optimised DNA encoding 6×His Aspergillus         indologenus glucosidase, Uniparc reference UPI000D7FE1ED,         Uniprot reference A0A2V5IY78.     -   Seq ID No. 525: Codon optimised DNA encoding 6×His Acaromyces         ingoldii glucosidase, Uniparc reference UPI000D802B25, Uniprot         reference A0A316YR39.     -   Seq ID No. 526: Codon optimised DNA encoding 6×His Monilinia         fructigena glucosidase, Uniparc reference UPI000DC60823, Uniprot         reference A0A395J1U5.     -   Seq ID No. 527: Codon optimised DNA encoding 6×His Paenibacillus         xylanexedens glucosidase, Uniparc reference UPI000F52D773,         Uniprot reference A0A3N6CA02.     -   Seq ID No. 528: Codon optimised DNA encoding 6×His Actinomyces         howellii glucosidase, Uniparc reference UPI000F6DAAAE, Uniprot         reference A0A448HIG0.     -   Seq ID No. 529: Codon optimised DNA encoding 6×His         Friedmanniomyces endolithicus glucosidase, Uniparc reference         UPI00113D19DF, Uniprot reference A0A4V5N9I4.     -   Seq ID No. 530: Codon optimised DNA encoding 6×His Neosartorya         fischeri glucosidase, Uniparc reference UPI0000EA8672, Uniprot         reference A1DNS0.     -   Seq ID No. 531: Codon optimised DNA encoding 6×His Pseudomonas         aeruginosa glucosidase, Uniparc reference UPI0000D7314B, Uniprot         reference A6V4K6.     -   Seq ID No. 532: Codon optimised DNA encoding 6×His Talaromyces         stipitatus glucosidase, Uniparc reference UPI00018E7266, Uniprot         reference B8MF24.     -   Seq ID No. 533: Codon optimised DNA encoding 6×His Aquimixticola         soesokkakensis glucosidase, Uniparc reference UPI000A1A5FD7,         Uniprot reference A0A1Y5RVF8.     -   Seq ID No. 534: Codon optimised DNA encoding 6×His         Rhodobacterales bacterium glucosidase, Uniparc reference         UPI000C98D37C, Uniprot reference A0A2D9YGV1.     -   Seq ID No. 535: Codon optimised DNA encoding 6×His Methylorubrum         extorquens glucosidase, Uniparc reference UPI0006F9793E, Uniprot         reference A0A2N9AS40.     -   Seq ID No. 536: Codon optimised DNA encoding 6×His Clostridium         perfringens glucosidase, Uniparc reference UPI00000CF7C8,         Uniprot reference A0A2X2YBP0.     -   Seq ID No. 537: Codon optimised DNA encoding 6×His         Acholeplasmatales bacterium glucosidase, Uniparc reference         UPI0008AEBEA3, Uniprot reference A0A348NID6.     -   Seq ID No. 538: Codon optimised DNA encoding 6×His Bacteroidetes         bacterium glucosidase, Uniparc reference UPI000EC3C979, Uniprot         reference A0A3B8VHE8.     -   Seq ID No. 539: Codon optimised DNA encoding 6×His Clostridium         carnis glucosidase, Uniparc reference UPI000F637E38, Uniprot         reference A0A3P6K8E8.     -   Seq ID No. 540: Codon optimised DNA encoding 6×His         Mycolicibacterium flavescens glucosidase, Uniparc reference         UPI000B93B5C9, Uniprot reference A0A448HNB5.     -   Seq ID No. 541: Codon optimised DNA encoding 6×His Streptococcus         gallolyticus glucosidase, Uniparc reference UPI000F6EFA71,         Uniprot reference A0A4V6LJ94.     -   Seq ID No. 542: Codon optimised DNA encoding 6×His Yersinia         enterocolitica glucosidase, Uniparc reference UPI0000EB54CC,         Uniprot reference A1JNB7.     -   Seq ID No. 543: Codon optimised DNA encoding 6×His         Anaeromyxobacter sp. glucosidase, Uniparc reference         UPI0000ED8A80, Uniprot reference A7HFG4.     -   Seq ID No. 544: Codon optimised DNA encoding 6×His Talaromyces         stipitatus glucosidase, Uniparc reference UPI00018E7D70, Uniprot         reference B8MK55.     -   Seq ID No. 545: Codon optimised DNA encoding 6×His Hortaea         werneckii glucosidase, Uniparc reference UPI000A2E3FAA, Uniprot         reference A0A1Z5SL14.     -   Seq ID No. 546: Codon optimised DNA encoding 6×His Micavibrio         sp. glucosidase, Uniparc reference UPI000C529025, Uniprot         reference A0A2E2Q8X2.     -   Seq ID No. 547: Codon optimised DNA encoding 6×His         Acidobacteriia bacterium glucosidase, Uniparc reference         UPI000CE6B996, Uniprot reference A0A2N9MBS0.     -   Seq ID No. 548: Codon optimised DNA encoding 6×His         Corynebacterium jeikeium glucosidase, Uniparc reference         UPI000DA3A972, Uniprot reference A0A2X4T570.     -   Seq ID No. 549: Codon optimised DNA encoding 6×His         Clostridiaceae bacterium glucosidase, Uniparc reference         UPI000E8D37A1, Uniprot reference A0A353PZH8.     -   Seq ID No. 550: Codon optimised DNA encoding 6×His         Anaerolineaceae bacterium glucosidase, Uniparc reference         UPI000748C096, Uniprot reference A0A3B9PA35.     -   Seq ID No. 551: Codon optimised DNA encoding 6×His Gymnopilus         dilepis glucosidase, Uniparc reference UPI000FF41956, Uniprot         reference A0A409WSY0.     -   Seq ID No. 552: Codon optimised DNA encoding 6×His Kocuria rosea         glucosidase, Uniparc reference UPI000F7105D4, Uniprot reference         A0A448R8N0.     -   Seq ID No. 553: Codon optimised DNA encoding 6×His         Teredinibacter sp. glucosidase, Uniparc reference UPI0011696FAB,         Uniprot reference A0A509DWZ3.     -   Seq ID No. 554: Codon optimised DNA encoding 6×His Aspergillus         niger glucosidase, Uniparc reference UPI0000EFB564, Uniprot         reference A2QS42.     -   Seq ID No. 555: Codon optimised DNA encoding 6×His Laccaria         bicolor glucosidase, Uniparc reference UPI000164423D, Uniprot         reference B0D734.     -   Seq ID No. 556: Codon optimised DNA encoding 6×His Podosphaera         parvula glucosidase, Uniparc reference UPI00017357F6, Uniprot         reference B9XH33.     -   Seq ID No. 557: Codon optimised DNA encoding 6×His Megamonas         hypermegale glucosidase, Uniparc reference UPI00042469F6,         Uniprot reference A0A239TGH2.     -   Seq ID No. 558: Codon optimised DNA encoding 6×His Armillaria         gallica glucosidase, Uniparc reference UPI000BC209C3, Uniprot         reference A0A2H3E300.     -   Seq ID No. 559: Codon optimised DNA encoding 6×His         Micromonospora sp. glucosidase, Uniparc reference UPI000D2EAE87,         Uniprot reference A0A2P8AV03.     -   Seq ID No. 560: Codon optimised DNA encoding 6×His Klebsiella         oxytoca glucosidase, Uniparc reference UPI000DA286EE, Uniprot         reference A0A2X5CJC5.     -   Seq ID No. 561: Codon optimised DNA encoding 6×His Candidatus         ozemobacter glucosidase, Uniparc reference UPI000DFAEF6C,         Uniprot reference A0A367ZIH8.     -   Seq ID No. 562: Codon optimised DNA encoding 6×His Coleophoma         crateriformis glucosidase, Uniparc reference UPI000E399EE8,         Uniprot reference A0A3D8R2C2.     -   Seq ID No. 563: Codon optimised DNA encoding 6×His Apiotrichum         porosum glucosidase, Uniparc reference UPI000FBC01E3, Uniprot         reference A0A427XHS2.     -   Seq ID No. 564: Codon optimised DNA encoding 6×His Acholeplasma         hippikon glucosidase, Uniparc reference UPI00068E4E50, Uniprot         reference A0A449BJ27.     -   Seq ID No. 565: Codon optimised DNA encoding 6×His Streptomyces         spectabilis glucosidase, Uniparc reference UPI001185F074,         Uniprot reference A0A516RGT1.     -   Seq ID No. 566: Codon optimised DNA encoding 6×His Aspergillus         niger glucosidase, Uniparc reference UPI0000EFCED2, Uniprot         reference A2R8G2.     -   Seq ID No. 567: Codon optimised DNA encoding 6×His Xanthomonas         campestris glucosidase, Uniparc reference UPI00000D8BFA, Uniprot         reference B0RYA0.     -   Seq ID No. 568: Codon optimised DNA encoding 6×His Lactobacillus         paracasei glucosidase, Uniparc reference UPI00019C9CD7, Uniprot         reference C2FDL2.     -   Seq ID No. 569: Codon optimised DNA encoding 6×His Bifiguratus         adelaidae glucosidase, Uniparc reference UPI000BC490A3, Uniprot         reference A0A261XUH4.     -   Seq ID No. 570: Codon optimised DNA encoding 6×His bacterium         glucosidase, Uniparc reference UPI000CC31AE2, Uniprot reference         A0A2H5YYA1.     -   Seq ID No. 571: Codon optimised DNA encoding 6×His Actinomadura         parvosata glucosidase, Uniparc reference UPI000D26C9C6, Uniprot         reference A0A2P9IY35.     -   Seq ID No. 572: Codon optimised DNA encoding 6×His Melissococcus         plutonius glucosidase, Uniparc reference UPI00024F22B8, Uniprot         reference A0A2Z5Y4P3.     -   Seq ID No. 573: Codon optimised DNA encoding 6×His Enterococcus         durans glucosidase, Uniparc reference UPI000E020871, Uniprot         reference A0A377KJS3.     -   Seq ID No. 574: Codon optimised DNA encoding 6×His Malassezia         restricta glucosidase, Uniparc reference UPI000DD17A7D, Uniprot         reference A0A3G2S2J6.     -   Seq ID No. 575: Codon optimised DNA encoding 6×His Apiotrichum         porosum glucosidase, Uniparc reference UPI000FA2DE87, Uniprot         reference A0A427XZQ0.     -   Seq ID No. 576: Codon optimised DNA encoding 6×His         Streptomonospora sp. glucosidase, Uniparc reference         UPI0010355193, Uniprot reference A0A4P6PWP5.     -   Seq ID No. 577: Codon optimised DNA encoding 6×His Lactobacillus         gasseri glucosidase, Uniparc reference UPI00119640A1, Uniprot         reference A0A558LH47.     -   Seq ID No. 578: Codon optimised DNA encoding 6×His Aspergillus         niger glucosidase, Uniparc reference UPI0000EFD0AA, Uniprot         reference A2RAJ1.     -   Seq ID No. 579: Codon optimised DNA encoding 6×His Neosartorya         fumigata glucosidase, Uniparc reference UPI000170BF91, Uniprot         reference B0XXG1.     -   Seq ID No. 580: Codon optimised DNA encoding 6×His Eubacterium         eligens glucosidase, Uniparc reference UPI0001A5B234, Uniprot         reference C4Z6T5.     -   Seq ID No. 581: Codon optimised DNA encoding 6×His Bifiguratus         adelaidae glucosidase, Uniparc reference UPI000BC64A75, Uniprot         reference A0A261XVM8.     -   Seq ID No. 582: Codon optimised DNA encoding 6×His bacterium         glucosidase, Uniparc reference UPI000CAA854E, Uniprot reference         A0A2H6EX57.     -   Seq ID No. 583: Codon optimised DNA encoding 6×His Corynespora         cassiicola glucosidase, Uniparc reference UPI000D24EC2C, Uniprot         reference A0A2T2N5H9.     -   Seq ID No. 584: Codon optimised DNA encoding 6×His         Pseudomicrostroma glucosiphilum glucosidase, Uniparc reference         UPI000D77A0B7, Uniprot reference A0A316UK83.     -   Seq ID No. 585: Codon optimised DNA encoding 6×His         Staphylococcus saprophyticus glucosidase, Uniparc reference         UPI0002DAAFCC, Uniprot reference A0A380HD31.     -   Seq ID No. 586: Codon optimised DNA encoding 6×His Malassezia         restricta glucosidase, Uniparc reference UPI000F0C6B8E, Uniprot         reference A0A3G2S932.     -   Seq ID No. 587: Codon optimised DNA encoding 6×His Saitozyma         podzolica glucosidase, Uniparc reference UPI000FBA70E2, Uniprot         reference A0A427YCL1.     -   Seq ID No. 588: Codon optimised DNA encoding 6×His Tremella         mesenterica glucosidase, Uniparc reference UPI00102822B4,         Uniprot reference A0A4Q1BRC6.     -   Seq ID No. 589: Codon optimised DNA encoding 6×His Arthrobacter         sp. glucosidase, Uniparc reference UPI0000527506, Uniprot         reference A0JZ86.     -   Seq ID No. 590: Codon optimised DNA encoding 6×His         Scheffersomyces stipitis glucosidase, Uniparc reference         UPI000157388C, Uniprot reference A3LRB0.     -   Seq ID No. 591: Codon optimised DNA encoding 6×His Leptothrix         cholodnii glucosidase, Uniparc reference UPI0001712E02, Uniprot         reference B1XZK8.     -   Seq ID No. 592: Codon optimised DNA encoding 6×His Thauera sp.         glucosidase, Uniparc reference UPI000166883C, Uniprot reference         C4ZLL7.     -   Seq ID No. 593: Codon optimised DNA encoding 6×His Kosmotoga         olearia glucosidase, Uniparc reference UPI00018494AB, Uniprot         reference C5CDW5.     -   Seq ID No. 594: Codon optimised DNA encoding 6×His Roseburia         intestinalis glucosidase, Uniparc reference UPI0001CD671F,         Uniprot reference D4L3Y2.     -   Seq ID No. 595: Codon optimised DNA encoding 6×His Streptococcus         equinus glucosidase, Uniparc reference UPI0001E0DC00, Uniprot         reference E0PDF8.     -   Seq ID No. 596: Codon optimised DNA encoding 6×His Streptococcus         cristatus glucosidase, Uniparc reference UPI0001F80C4B, Uniprot         reference E8JUK5.     -   Seq ID No. 597: Codon optimised DNA encoding 6×His         Cellulosilyticum lentocellum glucosidase, Uniparc reference         UPI0001D2DBBC, Uniprot reference F2JLH3.     -   Seq ID No. 598: Codon optimised DNA encoding 6×His Streptococcus         gallolyticus glucosidase, Uniparc reference UPI0001C48657,         Uniprot reference F5WYI5.     -   Seq ID No. 599: Codon optimised DNA encoding 6×His         Ketogulonicigenium vulgare glucosidase, Uniparc reference         UPI00021D443A, Uniprot reference F9Y8Z7.     -   Seq ID No. 600: Codon optimised DNA encoding 6×His Spathaspora         passalidarum glucosidase, Uniparc reference UPI000228255D,         Uniprot reference G3AGX1.     -   Seq ID No. 601: Codon optimised DNA encoding 6×His Niastella         koreensis glucosidase, Uniparc reference UPI00023F6F5F, Uniprot         reference G8T9J3.     -   Seq ID No. 602: Codon optimised DNA encoding 6×His Cellvibrio         sp. glucosidase, Uniparc reference UPI000260108C, Uniprot         reference I3IDC0.     -   Seq ID No. 603: Codon optimised DNA encoding 6×His         Flavobacterium sp. glucosidase, Uniparc reference UPI000272D1E0,         Uniprot reference J1ACA0.     -   Seq ID No. 604: Codon optimised DNA encoding 6×His Macrophomina         phaseolina glucosidase, Uniparc reference UPI00028E7FE1, Uniprot         reference K2S5D3.     -   Seq ID No. 605: Codon optimised DNA encoding 6×His Kosmotoga         olearia glucosidase, Uniparc reference UPI00018483A2, Uniprot         reference C5CHI5.     -   Seq ID No. 606: Codon optimised DNA encoding 6×His Blautia obeum         glucosidase, Uniparc reference UPI0001CD5918, Uniprot reference         D4LRF6.     -   Seq ID No. 607: Codon optimised DNA encoding 6×His         Bifidobacterium dentium glucosidase, Uniparc reference         UPI0001E18CDA, Uniprot reference E0Q541.     -   Seq ID No. 608: Codon optimised DNA encoding 6×His Anaerolinea         thermophila glucosidase, Uniparc reference UPI0001F55F8F,         Uniprot reference E8N5R8.     -   Seq ID No. 609: Codon optimised DNA encoding 6×His         Coriobacterium glomerans glucosidase, Uniparc reference         UPI0002050DA2, Uniprot reference F2N7E4.     -   Seq ID No. 610: Codon optimised DNA encoding 6×His Microlunatus         phosphovorus glucosidase, Uniparc reference UPI000210C886,         Uniprot reference F5XJQ3.     -   Seq ID No. 611: Codon optimised DNA encoding 6×His Streptomyces         sp. glucosidase, Uniparc reference UPI0001C18877, Uniprot         reference G0Q1U8.     -   Seq ID No. 612: Codon optimised DNA encoding 6×His Spathaspora         passalidarum glucosidase, Uniparc reference UPI0002282B61,         Uniprot reference G3AIV6.     -   Seq ID No. 613: Codon optimised DNA encoding 6×His Glarea         lozoyensis glucosidase, Uniparc reference UPI0002402F4A, Uniprot         reference H0ET34.     -   Seq ID No. 614: Codon optimised DNA encoding 6×His Glaciozyma         antarctica glucosidase, Uniparc reference UPI0002633B36, Uniprot         reference I3UJK0.     -   Seq ID No. 615: Codon optimised DNA encoding 6×His Arthrobacter         sp. glucosidase, Uniparc reference UPI00027DFD35, Uniprot         reference J7LN00.     -   Seq ID No. 616: Codon optimised DNA encoding 6×His Fusarium         pseudograminearum glucosidase, Uniparc reference UPI00028D698C,         Uniprot reference K3VMA9.     -   Seq ID No. 617: Codon optimised DNA encoding 6×His Hypocrea rufa         glucosidase, Uniparc reference UPI000006AA61, Uniprot reference         C6GGC9.     -   Seq ID No. 618: Codon optimised DNA encoding 6×His Ruminococcus         torques glucosidase, Uniparc reference UPI0001CDA964, Uniprot         reference D4M6W6.     -   Seq ID No. 619: Codon optimised DNA encoding 6×His         Bifidobacterium dentium glucosidase, Uniparc reference         UPI0001E17370, Uniprot reference E0Q9Z7.     -   Seq ID No. 620: Codon optimised DNA encoding 6×His Bacteroides         salanitronis glucosidase, Uniparc reference UPI0001FC71F6,         Uniprot reference F0R2D7.     -   Seq ID No. 621: Codon optimised DNA encoding 6×His Bacteroides         coprosuis glucosidase, Uniparc reference UPI00020E6D19, Uniprot         reference F3ZQ40.     -   Seq ID No. 622: Codon optimised DNA encoding 6×His Marinomonas         posidonica glucosidase, Uniparc reference UPI00020D4CC6, Uniprot         reference F6CWF6.     -   Seq ID No. 623: Codon optimised DNA encoding 6×His Chaetomium         thermophilum glucosidase, Uniparc reference UPI000227E8ED,         Uniprot reference G0SE64.     -   Seq ID No. 624: Codon optimised DNA encoding 6×His         Tetragenococcus halophilus glucosidase, Uniparc reference         UPI00022B9A15, Uniprot reference G4L5K1.     -   Seq ID No. 625: Codon optimised DNA encoding 6×His Paenibacillus         sp. glucosidase, Uniparc reference UPI00024F0867, Uniprot         reference H6CCZ8.     -   Seq ID No. 626: Codon optimised DNA encoding 6×His Turneriella         parva glucosidase, Uniparc reference UPI000265AA4B, Uniprot         reference 14B8U7.     -   Seq ID No. 627: Codon optimised DNA encoding 6×His Arthrobacter         sp. glucosidase, Uniparc reference UPI00027DFDB2, Uniprot         reference J7LQK9.     -   Seq ID No. 628: Codon optimised DNA encoding 6×His Agaricus         bisporus glucosidase, Uniparc reference UPI00029074B1, Uniprot         reference K5W7V1.     -   Seq ID No. 629: Codon optimised DNA encoding 6×His Nectria         haematococca glucosidase, Uniparc reference UPI0001B67634,         Uniprot reference C7YIP3.     -   Seq ID No. 630: Codon optimised DNA encoding 6×His Bacteroides         xylanisolvens glucosidase, Uniparc reference UPI0001A25287,         Uniprot reference D4VSZ0.     -   Seq ID No. 631: Codon optimised DNA encoding 6×His         Sediminispirochaeta smaragdinae glucosidase, Uniparc reference         UPI0001DD9790, Uniprot reference E1R331.     -   Seq ID No. 632: Codon optimised DNA encoding 6×His Deinococcus         proteolyticus glucosidase, Uniparc reference UPI0001FC42E9,         Uniprot reference F0RPV2.     -   Seq ID No. 633: Codon optimised DNA encoding 6×His         Sphingobacterium sp. glucosidase, Uniparc reference         UPI0002033A0A, Uniprot reference F4C226.     -   Seq ID No. 634: Codon optimised DNA encoding 6×His Sphingobium         chlorophenolicum glucosidase, Uniparc reference UPI0001E54133,         Uniprot reference F6ET40.     -   Seq ID No. 635: Codon optimised DNA encoding 6×His Caloramator         australicus glucosidase, Uniparc reference UPI00021CACC4,         Uniprot reference G0V3V5.     -   Seq ID No. 636: Codon optimised DNA encoding 6×His         Commensalibacter intestini glucosidase, Uniparc reference         UPI000230E3BF, Uniprot reference G6F370.     -   Seq ID No. 637: Codon optimised DNA encoding 6×His Paenibacillus         sp. glucosidase, Uniparc reference UPI00024F07AC, Uniprot         reference H6CIT2.     -   Seq ID No. 638: Codon optimised DNA encoding 6×His Nitrolancea         hollandica glucosidase, Uniparc reference UPI0002638AF3, Uniprot         reference I4EIA9.     -   Seq ID No. 639: Codon optimised DNA encoding 6×His Cryptococcus         neoformans glucosidase, Uniparc reference UPI000392C3ED, Uniprot         reference J9VVK7.     -   Seq ID No. 640: Codon optimised DNA encoding 6×His         Acidipropionibacterium acidipropionici glucosidase, Uniparc         reference UPI0002988588, Uniprot reference K75596.     -   Seq ID No. 641: Codon optimised DNA encoding 6×His Prevotella         sp. glucosidase, Uniparc reference UPI0001B93465, Uniprot         reference C9PT75.     -   Seq ID No. 642: Codon optimised DNA encoding 6×His Rhodobacter         capsulatus glucosidase, Uniparc reference UPI0001D08095, Uniprot         reference D5ALU0.     -   Seq ID No. 643: Codon optimised DNA encoding 6×His Stigmatella         aurantiaca glucosidase, Uniparc reference UPI0001E74370, Uniprot         reference E3FJ05.     -   Seq ID No. 644: Codon optimised DNA encoding 6×His Sphaerochaeta         globosa glucosidase, Uniparc reference UPI0002010060, Uniprot         reference F0RVK3.     -   Seq ID No. 645: Codon optimised DNA encoding 6×His Sphaerochaeta         coccoides glucosidase, Uniparc reference UPI000207D78C, Uniprot         reference F4GH96.     -   Seq ID No. 646: Codon optimised DNA encoding 6×His         Novosphingobium sp. glucosidase, Uniparc reference         UPI00020EFBCB, Uniprot reference F6ICQ5.     -   Seq ID No. 647: Codon optimised DNA encoding 6×His Arthrobotrys         oligospora glucosidase, Uniparc reference UPI000225331F, Uniprot         reference G1XH86.     -   Seq ID No. 648: Codon optimised DNA encoding 6×His Lactococcus         lactis glucosidase, Uniparc reference UPI00000C6907, Uniprot         reference G6FFS4.     -   Seq ID No. 649: Codon optimised DNA encoding 6×His         Phaeospirillum molischianum glucosidase, Uniparc reference         UPI000255313A, Uniprot reference H8FXH7.     -   Seq ID No. 650: Codon optimised DNA encoding 6×His Modestobacter         marinus glucosidase, Uniparc reference UPI0002609C68, Uniprot         reference I4EW72.     -   Seq ID No. 651: Codon optimised DNA encoding 6×His Saccharothrix         espanaensis glucosidase, Uniparc reference UPI00028AC337,         Uniprot reference K0K125.     -   Seq ID No. 652: Codon optimised DNA encoding 6×His Cronobacter         sakazakii glucosidase, Uniparc reference UPI00029BA293, Uniprot         reference K8DAK0.     -   Seq ID No. 653: Codon optimised DNA encoding 6×His Verticillium         alfalfae glucosidase, Uniparc reference UPI0001BBDF1E, Uniprot         reference C9SVX1.     -   Seq ID No. 654: Codon optimised DNA encoding 6×His Bacteroides         xylanisolvens glucosidase, Uniparc reference UPI0001CCFD36,         Uniprot reference D6CY10.     -   Seq ID No. 655: Codon optimised DNA encoding 6×His         Leadbetterella byssophila glucosidase, Uniparc reference         UPI0001EBD98A, Uniprot reference E4RUR0.     -   Seq ID No. 656: Codon optimised DNA encoding 6×His Sphaerochaeta         globosa glucosidase, Uniparc reference UPI00020102A5, Uniprot         reference F0RYB6.     -   Seq ID No. 657: Codon optimised DNA encoding 6×His Sphaerochaeta         coccoides glucosidase, Uniparc reference UPI000207D6A0, Uniprot         reference F4GLH6.     -   Seq ID No. 658: Codon optimised DNA encoding 6×His Haloplasma         contractile glucosidase, Uniparc reference UPI000212252C,         Uniprot reference F7Q0Y2.     -   Seq ID No. 659: Codon optimised DNA encoding 6×His         Nitrospirillum amazonense glucosidase, Uniparc reference         UPI0002265447, Uniprot reference G1Y407.     -   Seq ID No. 660: Codon optimised DNA encoding 6×His Azospirillum         brasilense glucosidase, Uniparc reference UPI00023420B8, Uniprot         reference G8AWD9.     -   Seq ID No. 661: Codon optimised DNA encoding 6×His         Phaeospirillum molischianum glucosidase, Uniparc reference         UPI000255314F, Uniprot reference H8FXJ8.     -   Seq ID No. 662: Codon optimised DNA encoding 6×His Modestobacter         marinus glucosidase, Uniparc reference UPI000260A2FA, Uniprot         reference I4EYD5.     -   Seq ID No. 663: Codon optimised DNA encoding 6×His         Wickerhamomyces ciferrii glucosidase, Uniparc reference         UPI000283EB8D, Uniprot reference K0KVJ2.     -   Seq ID No. 664: Codon optimised DNA encoding 6×His Gloeocapsa         sp. glucosidase, Uniparc reference UPI0002A5D085, Uniprot         reference K9XKL8.     -   Seq ID No. 665: Codon optimised DNA encoding 6×His Sphaerobacter         thermophilus glucosidase, Uniparc reference UPI0001A3BCB6,         Uniprot reference D1C7U8.     -   Seq ID No. 666: Codon optimised DNA encoding 6×His Bacteroides         xylanisolvens glucosidase, Uniparc reference UPI0001CCEF1F,         Uniprot reference D6D4V2.     -   Seq ID No. 667: Codon optimised DNA encoding 6×His Prevotella         buccae glucosidase, Uniparc reference UPI0001F149E8, Uniprot         reference E6K4W5.     -   Seq ID No. 668: Codon optimised DNA encoding 6×His Grosmannia         clavigera glucosidase, Uniparc reference UPI0001FF1101, Uniprot         reference F0XBR0.     -   Seq ID No. 669: Codon optimised DNA encoding 6×His Melampsora         larici-populina glucosidase, Uniparc reference UPI00020F9774,         Uniprot reference F4R4W2.     -   Seq ID No. 670: Codon optimised DNA encoding 6×His Prevotella         multisaccharivorax glucosidase, Uniparc reference UPI0002138E9C,         Uniprot reference F8N7G1.     -   Seq ID No. 671: Codon optimised DNA encoding 6×His Streptomyces         zinciresistens glucosidase, Uniparc reference UPI0002255A63,         Uniprot reference G2G8K4.     -   Seq ID No. 672: Codon optimised DNA encoding 6×His Granulicella         mallensis glucosidase, Uniparc reference UPI0001D9FC40, Uniprot         reference G8NY42.     -   Seq ID No. 673: Codon optimised DNA encoding 6×His Gibberella         zeae glucosidase, Uniparc reference UPI00021F1FEE, Uniprot         reference I1RH94.     -   Seq ID No. 674: Codon optimised DNA encoding 6×His Modestobacter         marinus glucosidase, Uniparc reference UPI00026090A2, Uniprot         reference I4EYK6.     -   Seq ID No. 675: Codon optimised DNA encoding 6×His Lactobacillus         equicursoris glucosidase, Uniparc reference UPI0002869F55,         Uniprot reference K0NRS8.     -   Seq ID No. 676: Codon optimised DNA encoding 6×His         Colletotrichum fructicola glucosidase, Uniparc reference         UPI0002A93280, Uniprot reference L2F9W0.     -   Seq ID No. 677: Codon optimised DNA encoding 6×His         Streptosporangium roseum glucosidase, Uniparc reference         UPI0001BF8AF6, Uniprot reference D2B261.     -   Seq ID No. 678: Codon optimised DNA encoding 6×His Listeria         grayi glucosidase, Uniparc reference UPI00019F252A, Uniprot         reference D7UX19.     -   Seq ID No. 679: Codon optimised DNA encoding 6×His Enterococcus         italicus glucosidase, Uniparc reference UPI0001F11EFF, Uniprot         reference E6LF07.     -   Seq ID No. 680: Codon optimised DNA encoding 6×His Fluviicola         taffensis glucosidase, Uniparc reference UPI000203D9EA, Uniprot         reference F2IIT6.     -   Seq ID No. 681: Codon optimised DNA encoding 6×His Shigella         flexneri glucosidase, Uniparc reference UPI00020CA641, Uniprot         reference F5N4W9.     -   Seq ID No. 682: Codon optimised DNA encoding 6×His Actinomyces         sp. glucosidase, Uniparc reference UPI0002189080, Uniprot         reference F9EFW5.     -   Seq ID No. 683: Codon optimised DNA encoding 6×His Verticillium         dahliae glucosidase, Uniparc reference UPI00022EBE5A, Uniprot         reference G2X5V6.     -   Seq ID No. 684: Codon optimised DNA encoding 6×His Actinoplanes         sp. glucosidase, Uniparc reference UPI00023ED5A8, Uniprot         reference G8S0M0.     -   Seq ID No. 685: Codon optimised DNA encoding 6×His Gibberella         zeae glucosidase, Uniparc reference UPI000023EA29, Uniprot         reference I1S320.     -   Seq ID No. 686: Codon optimised DNA encoding 6×His Auricularia         subglabra glucosidase, Uniparc reference UPI00027CE685, Uniprot         reference J0WV06.     -   Seq ID No. 687: Codon optimised DNA encoding 6×His         Nitratireductor indicus glucosidase, Uniparc reference         UPI00028E86D6, Uniprot reference K2N268.     -   Seq ID No. 688: Codon optimised DNA encoding 6×His         Thermoclostridium stercorarium glucosidase, Uniparc reference         UPI0002AD999E, Uniprot reference L7VH66.     -   Seq ID No. 689: Codon optimised DNA encoding 6×His Geobacillus         sp. glucosidase, Uniparc reference UPI0002AF2DE3, Uniprot         reference L8A1S2.     -   Seq ID No. 690: Codon optimised DNA encoding 6×His uncultured         bacterium glucosidase, Uniparc reference UPI000327C3CC, Uniprot         reference M9ZC55.     -   Seq ID No. 691: Codon optimised DNA encoding 6×His Burkholderia         ambifaria glucosidase, Uniparc reference UPI000059ACE1, Uniprot         reference Q0BAK2.     -   Seq ID No. 692: Codon optimised DNA encoding 6×His Aspergillus         oryzae glucosidase, Uniparc reference UPI0000676B8D, Uniprot         reference Q2UIR4.     -   Seq ID No. 693: Codon optimised DNA encoding 6×His Yersinia         pseudotuberculosis glucosidase, Uniparc reference UPI00004269B5,         Uniprot reference Q66DJ0.     -   Seq ID No. 694: Codon optimised DNA encoding 6×His Wallemia         ichthyophaga glucosidase, Uniparc reference UPI000331CC98,         Uniprot reference R9AF64.     -   Seq ID No. 695: Codon optimised DNA encoding 6×His Glarea         lozoyensis glucosidase, Uniparc reference UPI0003522404, Uniprot         reference S3DIM6.     -   Seq ID No. 696: Codon optimised DNA encoding 6×His         Moniliophthora roreri glucosidase, Uniparc reference         UPI0003BF753A, Uniprot reference V2YU31.     -   Seq ID No. 697: Codon optimised DNA encoding 6×His Zhouia         amylolytica glucosidase, Uniparc reference UPI0003DBCB4F,         Uniprot reference W2ULL3.     -   Seq ID No. 698: Codon optimised DNA encoding 6×His Fusarium         oxysporum glucosidase, Uniparc reference UPI0003F362FF, Uniprot         reference W9NQQ3.     -   Seq ID No. 699: Codon optimised DNA encoding 6×His         Flavobacterium johnsoniae glucosidase, Uniparc reference         UPI00006EA168, Uniprot reference A5FAA5.     -   Seq ID No. 700: Codon optimised DNA encoding 6×His Oryza sativa         glucosidase, Uniparc reference UPI00002394F5, Uniprot reference         Q75I93.     -   Seq ID No. 701: Codon optimised DNA encoding 6×His Clostridium         saccharoperbutylacetonicum glucosidase, Uniparc reference         UPI0002B65681, Uniprot reference M1MCD1.     -   Seq ID No. 702: Codon optimised DNA encoding 6×His Rhodococcus         sp. glucosidase, Uniparc reference UPI0002D21DB1, Uniprot         reference N1MBN6.     -   Seq ID No. 703: Codon optimised DNA encoding 6×His Burkholderia         ambifaria glucosidase, Uniparc reference UPI000059C0CD, Uniprot         reference Q0BCV8.     -   Seq ID No. 704: Codon optimised DNA encoding 6×His Xanthomonas         campestris glucosidase, Uniparc reference UPI00005CE9E7, Uniprot         reference Q3BVH7.     -   Seq ID No. 705: Codon optimised DNA encoding 6×His Caulobacter         vibrioides glucosidase, Uniparc reference UPI00000C7604, Uniprot         reference Q9A6F8.     -   Seq ID No. 706: Codon optimised DNA encoding 6×His Arcticibacter         svalbardensis glucosidase, Uniparc reference UPI000338772A,         Uniprot reference R9GRA7.     -   Seq ID No. 707: Codon optimised DNA encoding 6×His         Winogradskyella psychrotolerans glucosidase, Uniparc reference         UPI00035A4604, Uniprot reference S7VQ28.     -   Seq ID No. 708: Codon optimised DNA encoding 6×His         Methyloglobulus morosus glucosidase, Uniparc reference         UPI0003C4ED6A, Uniprot reference V5DXT8.     -   Seq ID No. 709: Codon optimised DNA encoding 6×His         Pestalotiopsis fici glucosidase, Uniparc reference         UPI0003E05C04, Uniprot reference W3WV37.     -   Seq ID No. 710: Codon optimised DNA encoding 6×His Capronia         coronata glucosidase, Uniparc reference UPI000434E698, Uniprot         reference W9YNR7.     -   Seq ID No. 711: Codon optimised DNA encoding 6×His Aspergillus         aculeatus glucosidase, Uniparc reference UPI00001268FD, Uniprot         reference P48825.     -   Seq ID No. 712: Codon optimised DNA encoding 6×His Thermotoga         neapolitana glucosidase, Uniparc reference UPI0000DD5996,         Uniprot reference Q0GC07.     -   Seq ID No. 713: Codon optimised DNA encoding 6×His Clostridium         saccharoperbutylacetonicum glucosidase, Uniparc reference         UPI0002B666E8, Uniprot reference M1MJF4.     -   Seq ID No. 714: Codon optimised DNA encoding 6×His         Schizosaccharomyces pombe glucosidase, Uniparc reference         UPI000006B001, Uniprot reference O74799.     -   Seq ID No. 715: Codon optimised DNA encoding 6×His Phaeosphaeria         nodorum glucosidase, Uniparc reference UPI000161BD2B, Uniprot         reference Q0TXF6.     -   Seq ID No. 716: Codon optimised DNA encoding 6×His Xylella         fastidiosa Dixon glucosidase, Uniparc reference UPI00003806C8,         Uniprot reference Q3RGJ3.     -   Seq ID No. 717: Codon optimised DNA encoding 6×His         Schizosaccharomyces pombe glucosidase, Uniparc reference         UPI000006A330, Uniprot reference Q9P6J6.     -   Seq ID No. 718: Codon optimised DNA encoding 6×His Arcticibacter         svalbardensis glucosidase, Uniparc reference UPI000337E887,         Uniprot reference R9GWD6.     -   Seq ID No. 719: Codon optimised DNA encoding 6×His         Colletotrichum gloeosporioides glucosidase, Uniparc reference         UPI0003885717, Uniprot reference T0KJI7.     -   Seq ID No. 720: Codon optimised DNA encoding 6×His uncultured         bacterium glucosidase, Uniparc reference UPI0003C9E340, Uniprot         reference V5R1E8.     -   Seq ID No. 721: Codon optimised DNA encoding 6×His Xanthomonas         arboricola glucosidase, Uniparc reference UPI0003E06A61, Uniprot         reference W4S7I5.     -   Seq ID No. 722: Codon optimised DNA encoding 6×His Fusarium         oxysporum glucosidase, Uniparc reference UPI00021EC697, Uniprot         reference X0A8X8.     -   Seq ID No. 723: Codon optimised DNA encoding 6×His Paenibacillus         polymyxa glucosidase, Uniparc reference UPI00001108D0, Uniprot         reference P22073.     -   Seq ID No. 724: Codon optimised DNA encoding 6×His Kluyveromyces         marxianus glucosidase, Uniparc reference UPI0001BE5ADA, Uniprot         reference D1GCC6.     -   Seq ID No. 725: Codon optimised DNA encoding 6×His Ilumatobacter         coccineus glucosidase, Uniparc reference UPI0002C04A25, Uniprot         reference M5A594.     -   Seq ID No. 726: Codon optimised DNA encoding 6×His Agrobacterium         sp. glucosidase, Uniparc reference UPI0000126912, Uniprot         reference P12614.     -   Seq ID No. 727: Codon optimised DNA encoding 6×His Cytophaga         hutchinsonii glucosidase, Uniparc reference UPI000038ECF7,         Uniprot reference Q11P53.     -   Seq ID No. 728: Codon optimised DNA encoding 6×His Thermobifida         fusca glucosidase, Uniparc reference UPI00003C5CA4, Uniprot         reference Q47PF5.     -   Seq ID No. 729: Codon optimised DNA encoding 6×His Botryotinia         fuckeliana glucosidase, Uniparc reference UPI0000069E5E, Uniprot         reference Q9UVJ6.     -   Seq ID No. 730: Codon optimised DNA encoding 6×His Agarivorans         albus glucosidase, Uniparc reference UPI00033994D2, Uniprot         reference R9PTK2.     -   Seq ID No. 731: Codon optimised DNA encoding 6×His Enterococcus         sp. glucosidase, Uniparc reference UPI00038B7639, Uniprot         reference T0UDU2.     -   Seq ID No. 732: Codon optimised DNA encoding 6×His Salinispira         pacifica glucosidase, Uniparc reference UPI0003D8A7DC, Uniprot         reference V5WKT4.     -   Seq ID No. 733: Codon optimised DNA encoding 6×His Bacteroides         xylanisolvens glucosidase, Uniparc reference UPI0003ECF15E,         Uniprot reference W6P696.     -   Seq ID No. 734: Codon optimised DNA encoding 6×His Fusarium         oxysporum glucosidase, Uniparc reference UPI000430029A, Uniprot         reference X0AB46.     -   Seq ID No. 735: Codon optimised DNA encoding 6×His Thermotoga         maritima glucosidase, Uniparc reference UPI0000126906, Uniprot         reference Q08638B.     -   Seq ID No. 736: Codon optimised DNA encoding 6×His Neotermes         koshunensis glucosidase, Uniparc reference UPI0000083EA8,         Uniprot reference Q8T0W7.     -   Seq ID No. 737: Codon optimised DNA encoding 6×His Thanatephorus         cucumeris glucosidase, Uniparc reference UPI0002BF34B3, Uniprot         reference M5CHG9.     -   Seq ID No. 738: Codon optimised DNA encoding 6×His         Hungateiclostridium thermocellum glucosidase, Uniparc reference         UPI000053581D, Uniprot reference P14002.     -   Seq ID No. 739: Codon optimised DNA encoding 6×His Koribacter         versatilis glucosidase, Uniparc reference UPI0000D76A70, Uniprot         reference Q1IJ89.     -   Seq ID No. 740: Codon optimised DNA encoding 6×His Neosartorya         fumigata glucosidase, Uniparc reference UPI00005203D8, Uniprot         reference Q4WLX5.     -   Seq ID No. 741: Codon optimised DNA encoding 6×His Amycolatopsis         vancoresmycina glucosidase, Uniparc reference UPI00032DB8C1,         Uniprot reference R1FKF0.     -   Seq ID No. 742: Codon optimised DNA encoding 6×His Gibberella         fujikuroi glucosidase, Uniparc reference UPI0003519C76, Uniprot         reference S0EKU1.     -   Seq ID No. 743: Codon optimised DNA encoding 6×His         Bifidobacterium longum glucosidase, Uniparc reference         UPI000390F839, Uniprot reference T2I2H5.     -   Seq ID No. 744: Codon optimised DNA encoding 6×His uncultured         bacterium glucosidase, Uniparc reference UPI0003DFF0DA, Uniprot         reference W0FLD1.     -   Seq ID No. 745: Codon optimised DNA encoding 6×His Bacteroides         xylanisolvens glucosidase, Uniparc reference UPI0002D3D994,         Uniprot reference W6P9J9.     -   Seq ID No. 746: Codon optimised DNA encoding 6×His Fusarium         oxysporum glucosidase, Uniparc reference UPI00042F8016, Uniprot         reference X0HA35.     -   Seq ID No. 747: Codon optimised DNA encoding 6×His Sorghum         bicolor glucosidase, Uniparc reference UPI00000A7F40, Uniprot         reference Q41290.     -   Seq ID No. 748: Codon optimised DNA encoding 6×His Clostridium         cellulovorans glucosidase, Uniparc reference UPI000050B701,         Uniprot reference Q53EH2.     -   Seq ID No. 749: Codon optimised DNA encoding 6×His Dacryopinax         primogenitus glucosidase, Uniparc reference UPI0002C29EAA,         Uniprot reference M5G1U5.     -   Seq ID No. 750: Codon optimised DNA encoding 6×His Rhizobium         radiobacter glucosidase, Uniparc reference UPI0000126913,         Uniprot reference P27034.     -   Seq ID No. 751: Codon optimised DNA encoding 6×His Phanerochaete         chrysosporium glucosidase, Uniparc reference UPI00006E0CC7,         Uniprot reference Q25BW5A.     -   Seq ID No. 752: Codon optimised DNA encoding 6×His Enterobacter         agglomerans glucosidase, Uniparc reference UPI0000126905,         Uniprot reference Q59437.     -   Seq ID No. 753: Codon optimised DNA encoding 6×His Candidatus         microthrix glucosidase, Uniparc reference UPI00033056C4, Uniprot         reference R4YWU4.     -   Seq ID No. 754: Codon optimised DNA encoding 6×His Chthonomonas         calidirosea glucosidase, Uniparc reference UPI0003427F29,         Uniprot reference S0ET37.     -   Seq ID No. 755: Codon optimised DNA encoding 6×His Acholeplasma         brassicae glucosidase, Uniparc reference UPI0003B04208, Uniprot         reference U4KMR7.     -   Seq ID No. 756: Codon optimised DNA encoding 6×His Chania         multitudinisentens glucosidase, Uniparc reference UPI0003E13BD3,         Uniprot reference W0LJ28.     -   Seq ID No. 757: Codon optimised DNA encoding 6×His Gibberella         moniliformis glucosidase, Uniparc reference UPI0003ECCF80,         Uniprot reference W7MW98.     -   Seq ID No. 758: Codon optimised DNA encoding 6×His Fusarium         oxysporum glucosidase, Uniparc reference UPI0004305856, Uniprot         reference X0I2U8.     -   Seq ID No. 759: Codon optimised DNA encoding 6×His Phanerochaete         chrysosporium glucosidase, Uniparc reference UPI00006E0CC7,         Uniprot reference Q25BW5B.     -   Seq ID No. 760: Codon optimised DNA encoding 6×His Secale         cereale glucosidase, Uniparc reference UPI00000A7EFC, Uniprot         reference Q9FYS3.     -   Seq ID No. 761: Codon optimised DNA encoding 6×His Anoxybacillus         gonensis glucosidase, Uniparc reference UPI0002C0273E, Uniprot         reference M5QUM2.     -   Seq ID No. 762: Codon optimised DNA encoding 6×His Thermotoga         maritima glucosidase, Uniparc reference UPI0000126906, Uniprot         reference Q08638A.     -   Seq ID No. 763: Codon optimised DNA encoding 6×His         Rhodospirillum rubrum glucosidase, Uniparc reference         UPI00003C2ACC, Uniprot reference Q2RP51.     -   Seq ID No. 764: Codon optimised DNA encoding 6×His Thermotoga         neapolitana glucosidase, Uniparc reference UPI00000B9013,         Uniprot reference Q60038.     -   Seq ID No. 765: Codon optimised DNA encoding 6×His Candidatus         microthrix glucosidase, Uniparc reference UPI00032F466E, Uniprot         reference R4Z6M6.     -   Seq ID No. 766: Codon optimised DNA encoding 6×His         Ruminiclostridium cellobioparum glucosidase, Uniparc reference         UPI00032874EC, Uniprot reference S0FPI8.     -   Seq ID No. 767: Codon optimised DNA encoding 6×His Acholeplasma         brassicae glucosidase, Uniparc reference UPI0003B0490C, Uniprot         reference U4KMV4.     -   Seq ID No. 768: Codon optimised DNA encoding 6×His Klebsiella         pneumoniae glucosidase, Uniparc reference UPI0003DB6EFD, Uniprot         reference W1BBP5.     -   Seq ID No. 769: Codon optimised DNA encoding 6×His Enterobacter         sp. glucosidase, Uniparc reference UPI0003ED0A8E, Uniprot         reference W7P6Y1.     -   Seq ID No. 770: Codon optimised DNA encoding 6×His Rhizoctonia         solani glucosidase, Uniparc reference UPI00045BB507, Uniprot         reference X8JI88.     -   Seq ID No. 771: Codon optimised DNA encoding 6×His Homo sapiens         glucosidase, Uniparc reference UPI0000072C73, Uniprot reference         Q9H227.     -   Seq ID No. 772: Codon optimised DNA encoding 6×His uncultured         bacterium glucosidase, Uniparc reference UPI0000DD5304, Uniprot         reference Q0GMU3.     -   Seq ID No. 773: Codon optimised DNA encoding 6×His         Thermoanaerobacter thermohydrosulfuricus glucosidase, Uniparc         reference UPI0002CA9E4F, Uniprot reference M8CQD9.     -   Seq ID No. 774: Codon optimised DNA encoding 6×His Stigmatella         aurantiaca glucosidase, Uniparc reference UPI0000E297D8, Uniprot         reference Q091M8.     -   Seq ID No. 775: Codon optimised DNA encoding 6×His Burkholderia         thailandensis glucosidase, Uniparc reference UPI00006676B1,         Uniprot reference Q2T7C9.     -   Seq ID No. 776: Codon optimised DNA encoding 6×His Yersinia         pseudotuberculosis glucosidase, Uniparc reference UPI0000426E8F,         Uniprot reference Q665S2.     -   Seq ID No. 777: Codon optimised DNA encoding 6×His Togninia         minima glucosidase, Uniparc reference UPI00032BF71F, Uniprot         reference R8BQM8.     -   Seq ID No. 778: Codon optimised DNA encoding 6×His Ophiostoma         piceae glucosidase, Uniparc reference UPI00035213C2, Uniprot         reference S3BVJ8.     -   Seq ID No. 779: Codon optimised DNA encoding 6×His Pyronema         omphalodes glucosidase, Uniparc reference UPI0003B117B7, Uniprot         reference U4LIL3.     -   Seq ID No. 780: Codon optimised DNA encoding 6×His Ogataea         parapolymorpha glucosidase, Uniparc reference UPI0001F76D1F,         Uniprot reference W1QIW0.     -   Seq ID No. 781: Codon optimised DNA encoding 6×His Hymenobacter         swuensis glucosidase, Uniparc reference UPI0003F2059F, Uniprot         reference W8F6U0.     -   Seq ID No. 782: Codon optimised DNA encoding 6×His         Flavobacterium johnsoniae glucosidase, Uniparc reference         UPI00006E8E1B, Uniprot reference A5FEF5.     -   Seq ID No. 783: Codon optimised DNA encoding 6×His Paenibacillus         polymyxa glucosidase, Uniparc reference UPI000012690B, Uniprot         reference P22505.     -   Seq ID No. 784: Codon optimised DNA encoding 6×His Oryza sativa         glucosidase, Uniparc reference UPI000009D014, Uniprot reference         Q8L7J2.     -   Seq ID No. 785: Codon optimised DNA encoding 6×His Oryza sativa         glucosidase, Uniparc reference UPI0000E580F2, Uniprot reference         B8AVF0.     -   Seq ID No. 786: Codon optimised DNA encoding 6×His Nannochloris         glucosidase, Uniparc reference UPI000EA7F16F, Uniprot reference         A0A452CSM4.     -   Seq ID No. 787: Codon optimised DNA encoding 6×His         Halothermothrix orenii glucosidase, Uniparc reference         UPI00006AE508, Uniprot reference B8CYA8.     -   Seq ID No. 788: Codon optimised DNA encoding 6×His Neurospora         crassa glucosidase, Uniparc reference UPI000018B2B4, Uniprot         reference Q7RWP2.     -   Seq ID No. 789: Codon optimised DNA encoding 6×His Micrococcus         antarcticus glucosidase, Uniparc reference UPI000192BB5F,         Uniprot reference B9V8P5.     -   Seq ID No. 790: Codon optimised DNA encoding 6×His         Exiguobacterium antarcticum glucosidase, Uniparc reference         UPI000285E79E, Uniprot reference K0A8J9.     -   Seq ID No. 791: Codon optimised DNA encoding 6×His Thermus         thermophilus glucosidase, Uniparc reference UPI00000BEB61,         Uniprot reference Q9RA61.     -   Seq ID No. 792: Codon optimised DNA encoding 6×His Trichoderma         harzianum glucosidase, Uniparc reference UPI00078BF747, Uniprot         reference A0A2T4AR08.     -   Seq ID No. 793: Codon optimised DNA encoding 6×His Hypocrea         jecorina glucosidase, Uniparc reference UPI000006AA61, Uniprot         reference Q12715.     -   Seq ID No. 794: Codon optimised DNA encoding 6×His Streptomyces         sp. glucosidase, Uniparc reference UPI00000B411B, Uniprot         reference Q59976.     -   Seq ID No. 795: Codon optimised DNA encoding 6×His Streptococcus         pyogenes glucosidase, Uniparc reference UPI00000C7E56, Uniprot         reference Q99YP9.     -   Seq ID No. 796: Codon optimised DNA encoding 6×His Trifolium         repens glucosidase, Uniparc reference UPI000012691B, Uniprot         reference P26205.     -   Seq ID No. 797: Codon optimised DNA encoding 6×His Talaromyces         emersonii glucosidase, Uniparc reference UPI000006C8FF, Uniprot         reference Q8TGI8.     -   Seq ID No. 798: Codon optimised DNA encoding 6×His         Hungateiclostridium thermocellum glucosidase, Uniparc reference         UPI0000126903, Uniprot reference P26208.     -   Seq ID No. 799: Codon optimised DNA encoding 6×His Lactobacillus         plantarum glucosidase, Uniparc reference UPI000219FE3E, Uniprot         reference F9ULH8.     -   Seq ID No. 800: Codon optimised DNA encoding 6×His Agrobacterium         tumefaciens glucosidase, Uniparc reference UPI0003F2033A,         Uniprot reference A0A2I4PGZ0.     -   Seq ID No. 801: Amino acid sequence for Bifidobacterium         actinocoloniiforme glucosidase, Uniparc reference UPI000503B26C,         Uniprot reference A0A086YYS8.     -   Seq ID No. 802: Amino acid sequence for Bifidobacterium         psychraerophilum glucosidase, Uniparc reference UPI000502B461,         Uniprot reference A0A087CD28.     -   Seq ID No. 803: Amino acid sequence for Penicillium italicum         glucosidase, Uniparc reference UPI00052B8681, Uniprot reference         A0A0A2K704.     -   Seq ID No. 804: Amino acid sequence for Microbacterium         trichothecenolyticum glucosidase, Uniparc reference         UPI0005ED0AE6, Uniprot reference A0A0M2HDB3.     -   Seq ID No. 805: Amino acid sequence for uncultured Clostridium         sp. glucosidase, Uniparc reference UPI0008206F38, Uniprot         reference A0A1C6I6A3.     -   Seq ID No. 806: Amino acid sequence for Bifiguratus adelaidae         glucosidase, Uniparc reference UPI000BC57319, Uniprot reference         A0A261Y7Q8.     -   Seq ID No. 807: Amino acid sequence for Paenibacillus         thiaminolyticus glucosidase, Uniparc reference UPI000B3B73CD,         Uniprot reference A0A378ZIK3.     -   Seq ID No. 808: Amino acid sequence for Microbacterium         lemovicicum glucosidase, Uniparc reference UPI000F8FB9BA,         Uniprot reference A0A3S9WE68.     -   Seq ID No. 809: Amino acid sequence for Rhodococcus erythropolis         glucosidase, Uniparc reference UPI00019923DC, Uniprot reference         C1A1N4.     -   Seq ID No. 810: Amino acid sequence for Cutibacterium avidum         glucosidase, Uniparc reference UPI00022C19B5, Uniprot reference         G4CZU8.     -   Seq ID No. 811: Amino acid sequence for Clavibacter         michiganensis glucosidase, Uniparc reference UPI0002C5A938,         Uniprot reference M5BD10.     -   Seq ID No. 8I2: Amino acid sequence for Microbacterium sp.         glucosidase, Uniparc reference UPI0003DE3509, Uniprot reference         W0ZC23.     -   Seq ID No. 813: Amino acid sequence for Bifidobacterium         actinocoloniiforme glucosidase, Uniparc reference UPI000507F38A,         Uniprot reference A0A086YZL4.     -   Seq ID No. 814: Amino acid sequence for Bifidobacterium reuteri         glucosidase, Uniparc reference UPI000506119A, Uniprot reference         A0A087CR26.     -   Seq ID No. 815: Amino acid sequence for Propionibacterium         freudenreichii glucosidase, Uniparc reference UPI0005A5CAC2,         Uniprot reference A0A0A8RX48.     -   Seq ID No. 816: Amino acid sequence for Microbacterium         hydrocarbonoxydans glucosidase, Uniparc reference UPI0005EC18F9,         Uniprot reference A0A0M2HRU0.     -   Seq ID No. 817: Amino acid sequence for Pseudonocardia sp.         glucosidase, Uniparc reference UPI00094B6D3B, Uniprot reference         A0A1Q8KSV8.     -   Seq ID No. 818: Amino acid sequence for Tuber aestivum         glucosidase, Uniparc reference UPI000BC13DF0, Uniprot reference         A0A292PKV5.     -   Seq ID No. 819: Amino acid sequence for Propionibacterium         australiense glucosidase, Uniparc reference UPI000E5B4EA5,         Uniprot reference A0A383S7A9.     -   Seq ID No. 820: Amino acid sequence for Fusarium sp.         glucosidase, Uniparc reference UPI001004B2C9, Uniprot reference         A0A428T6E2.     -   Seq ID No. 821: Amino acid sequence for Nectria haematococca         glucosidase, Uniparc reference UPI0001B69B5C, Uniprot reference         C7ZBV0.     -   Seq ID No. 822: Amino acid sequence for Actinoplanes sp.         glucosidase, Uniparc reference UPI00023EBB15, Uniprot reference         G8S3E7.     -   Seq ID No. 823: Amino acid sequence for Streptomyces fulvissimus         glucosidase, Uniparc reference UPI0003289BC6, Uniprot reference         N0CMW2.     -   Seq ID No. 824: Amino acid sequence for Pestalotiopsis fici         glucosidase, Uniparc reference UPI0003E02BF1, Uniprot reference         W3WXF1.     -   Seq ID No. 825: Amino acid sequence for Bifidobacterium         bohemicum glucosidase, Uniparc reference UPI0005025F2E, Uniprot         reference A0A086ZGP0.     -   Seq ID No. 826: Amino acid sequence for Bifidobacterium         saeculare glucosidase, Uniparc reference UPI0005084E52, Uniprot         reference A0A087D0Q2.     -   Seq ID No. 827: Amino acid sequence for Bionectria ochroleuca         glucosidase, Uniparc reference UPI00059674D6, Uniprot reference         A0A0B7JW24.     -   Seq ID No. 828: Amino acid sequence for Bifidobacterium         pseudocatenulatum glucosidase, Uniparc reference UPI0006C6D6F9,         Uniprot reference A0A174AU04.     -   Seq ID No. 829: Amino acid sequence for Pseudonocardia sp.         glucosidase, Uniparc reference UPI00094ABAB3, Uniprot reference         A0A1Q8LPB4.     -   Seq ID No. 830: Amino acid sequence for bacterium glucosidase,         Uniparc reference UPI000CB49A0B, Uniprot reference A0A2H5Z8Q4.     -   Seq ID No. 831: Amino acid sequence for Coleophoma crateriformis         glucosidase, Uniparc reference UPI000E38A995, Uniprot reference         A0A3D8Q771.     -   Seq ID No. 832: Amino acid sequence for Arthrobotrys oligospora         glucosidase, Uniparc reference UPI001102DFA3, Uniprot reference         A0A4Z0Y5Y8.     -   Seq ID No. 833: Amino acid sequence for Pyrenophora teres         glucosidase, Uniparc reference UPI0001ECDCBD, Uniprot reference         E3RFS2.     -   Seq ID No. 834: Amino acid sequence for Gordonia         polyisoprenivorans glucosidase, Uniparc reference UPI00024F2A26,         Uniprot reference H6MTQ7.     -   Seq ID No. 835: Amino acid sequence for Stigmatella aurantiaca         glucosidase, Uniparc reference UPI0000E28E5D, Uniprot reference         Q08521.     -   Seq ID No. 836: Amino acid sequence for Pestalotiopsis fici         glucosidase, Uniparc reference UPI0003E03A52, Uniprot reference         W3WZ03.     -   Seq ID No. 837: Amino acid sequence for Bifidobacterium magnum         glucosidase, Uniparc reference UPI0003B7B6EE, Uniprot reference         A0A087BEN9.     -   Seq ID No. 838: Amino acid sequence for Bifidobacterium         stellenboschense glucosidase, Uniparc reference UPI000503F283,         Uniprot reference A0A087DFL8.     -   Seq ID No. 839: Amino acid sequence for Bionectria ochroleuca         glucosidase, Uniparc reference UPI00059673EE, Uniprot reference         A0A0B7K316.     -   Seq ID No. 840: Amino acid sequence for Hungatella hathewayi         glucosidase, Uniparc reference UPI0006C069F3, Uniprot reference         A0A174LVE3.     -   Seq ID No. 841: Amino acid sequence for Mycetocola reblochoni         glucosidase, Uniparc reference UPI00097EB800, Uniprot reference         A0A1R4J2F9.     -   Seq ID No. 842: Amino acid sequence for Nonomuraea sp.         glucosidase, Uniparc reference UPI0009ABD7B3, Uniprot reference         A0A2P9IX34.     -   Seq ID No. 843: Amino acid sequence for Coleophoma crateriformis         glucosidase, Uniparc reference UPI000E391DF6, Uniprot reference         A0A3D8T9C2.     -   Seq ID No. 844: Amino acid sequence for Paenarthrobacter         aurescens glucosidase, Uniparc reference UPI0000EC83AE, Uniprot         reference A1R2K1.     -   Seq ID No. 845: Amino acid sequence for Kitasatospora setae         glucosidase, Uniparc reference UPI0001F21F08, Uniprot reference         E4N4F6.     -   Seq ID No. 846: Amino acid sequence for Nocardiopsis alba         glucosidase, Uniparc reference UPI00027E251E, Uniprot reference         J7L3Z8.     -   Seq ID No. 847: Amino acid sequence for Rhodococcus jostii         glucosidase, Uniparc reference UPI0000DBA338, Uniprot reference         Q0SCI4.     -   Seq ID No. 848: Amino acid sequence for uncultured microorganism         glucosidase, Uniparc reference UPI0003EC942D, Uniprot reference         W5X324.     -   Seq ID No. 849: Amino acid sequence for Bifidobacterium         merycicum glucosidase, Uniparc reference UPI0005084B63, Uniprot         reference A0A087BJ88.     -   Seq ID No. 850: Amino acid sequence for Bifidobacterium         scardovii glucosidase, Uniparc reference UPI0005018B5B, Uniprot         reference A0A087DGT3.     -   Seq ID No. 851: Amino acid sequence for Fusarium oxysporum         glucosidase, Uniparc reference UPI00021EC81E, Uniprot reference         A0A0C4DJL4.     -   Seq ID No. 852: Amino acid sequence for Paraphaeosphaeria         sporulosa glucosidase, Uniparc reference UPI0007CE199C, Uniprot         reference A0A177BXH1.     -   Seq ID No. 853: Amino acid sequence for Clostridium oryzae         glucosidase, Uniparc reference UPI0009A50F8D, Uniprot reference         A0A1V4IY77.     -   Seq ID No. 854: Amino acid sequence for Corynespora cassiicola         glucosidase, Uniparc reference UPI000D22F5EB, Uniprot reference         A0A2T2N4T6.     -   Seq ID No. 855: Amino acid sequence for Choiromyces venosus         glucosidase, Uniparc reference UPI000F7332F8, Uniprot reference         A0A3N4J9R6.     -   Seq ID No. 856: Amino acid sequence for Saccharopolyspora         erythraea glucosidase, Uniparc reference UPI0000F550EC, Uniprot         reference A4F982.     -   Seq ID No. 857: Amino acid sequence for Streptomyces venezuelae         glucosidase, Uniparc reference UPI000204906E, Uniprot reference         F2R0Y2.     -   Seq ID No. 858: Amino acid sequence for Arthrobacter sp.         glucosidase, Uniparc reference UPI00027DFDB0, Uniprot reference         J7LVB1.     -   Seq ID No. 859: Amino acid sequence for Thermobrachium celere         glucosidase, Uniparc reference UPI00033420CB, Uniprot reference         R7RMQ6.     -   Seq ID No. 860: Amino acid sequence for Drechslerella         stenobrocha glucosidase, Uniparc reference UPI0003EA65B9,         Uniprot reference W7HT93.     -   Seq ID No. 861: Amino acid sequence for Bifidobacterium minimum         glucosidase, Uniparc reference UPI0003B38542, Uniprot reference         A0A087BMR5.     -   Seq ID No. 862: Amino acid sequence for Bifidobacterium         thermacidophilum glucosidase, Uniparc reference UPI000409825E,         Uniprot reference A0A087E3K8.     -   Seq ID No. 863: Amino acid sequence for Verruconis gallopava         glucosidase, Uniparc reference UPI0005C0229F, Uniprot reference         A0A0D1X9G0.     -   Seq ID No. 864: Amino acid sequence for Stagonospora sp.         glucosidase, Uniparc reference UPI0007CECAD5, Uniprot reference         A0A178AER7.     -   Seq ID No. 865: Amino acid sequence for Firmicutes bacterium         glucosidase, Uniparc reference UPI0009D58C49, Uniprot reference         A0A1V6ALF9.     -   Seq ID No. 866: Amino acid sequence for Tuber borchii         glucosidase, Uniparc reference UPI000D50EED1, Uniprot reference         A0A2T6ZNU7.     -   Seq ID No. 867: Amino acid sequence for Morchella conica         glucosidase, Uniparc reference UPI000F728167, Uniprot reference         A0A3N4KUR3.     -   Seq ID No. 868: Amino acid sequence for Dictyoglomus         thermophilum glucosidase, Uniparc reference UPI00018152EB,         Uniprot reference B5YC96.     -   Seq ID No. 869: Amino acid sequence for Microlunatus         phosphovorus glucosidase, Uniparc reference UPI000210C01C,         Uniprot reference F5XL24.     -   Seq ID No. 870: Amino acid sequence for Bifidobacterium         asteroides glucosidase, Uniparc reference UPI00028BB1B6, Uniprot         reference K4IPD2.     -   Seq ID No. 871: Amino acid sequence for Dactylellina haptotyla         glucosidase, Uniparc reference UPI00035AE576, Uniprot reference         S8BQ60.     -   Seq ID No. 872: Amino acid sequence for Fusarium oxysporum         glucosidase, Uniparc reference UPI0003F2D7DD, Uniprot reference         W9JF54.     -   Seq ID No. 873: Amino acid sequence for Bifidobacterium longum         glucosidase, Uniparc reference UPI0005067FF9, Uniprot reference         A0A087BRY2.     -   Seq ID No. 874: Amino acid sequence for Bifidobacterium         tsurumiense glucosidase, Uniparc reference UPI0004105550,         Uniprot reference A0A087EK01.     -   Seq ID No. 875: Amino acid sequence for Exophiala spinifera         glucosidase, Uniparc reference UPI0005BF9DF9, Uniprot reference         A0A0D1YG50.     -   Seq ID No. 876: Amino acid sequence for Pyrenochaeta sp.         glucosidase, Uniparc reference UPI0007CE7DBA, Uniprot reference         A0A178E5I1.     -   Seq ID No. 877: Amino acid sequence for Firmicutes bacterium         glucosidase, Uniparc reference UPI0009CDA3F1, Uniprot reference         A0A1V6FLK2.     -   Seq ID No. 878: Amino acid sequence for Cadophora sp.         glucosidase, Uniparc reference UPI000D5A9E68, Uniprot reference         A0A2V1B599.     -   Seq ID No. 879: Amino acid sequence for Morchella conica         glucosidase, Uniparc reference UPI000F7330CD, Uniprot reference         A0A3N4L4M3.     -   Seq ID No. 880: Amino acid sequence for Bifidobacterium animalis         glucosidase, Uniparc reference UPI000189C68F, Uniprot reference         B8DV42.     -   Seq ID No. 881: Amino acid sequence for Treponema azotonutricium         glucosidase, Uniparc reference UPI00020ED2C9, Uniprot reference         F5YGD5.     -   Seq ID No. 882: Amino acid sequence for Acidipropionibacterium         acidipropionici glucosidase, Uniparc reference UPI0002988429,         Uniprot reference K7S0E5.     -   Seq ID No. 883: Amino acid sequence for Salinispira pacifica         glucosidase, Uniparc reference UPI0003D93613, Uniprot reference         V5WNB6.     -   Seq ID No. 884: Amino acid sequence for Bifidobacterium         mongoliense glucosidase, Uniparc reference UPI000503588E,         Uniprot reference A0A087C760.     -   Seq ID No. 885: Amino acid sequence for Bifidobacterium indicum         glucosidase, Uniparc reference UPI000499F5D1, Uniprot reference         A0A087VUW4.     -   Seq ID No. 886: Amino acid sequence for Brachyspira suanatina         glucosidase, Uniparc reference UPI000659257F, Uniprot reference         A0A0G4K5C2.     -   Seq ID No. 887: Amino acid sequence for Phialocephala         scopiformis glucosidase, Uniparc reference UPI0007F2FD44,         Uniprot reference A0A194X8Q9.     -   Seq ID No. 888: Amino acid sequence for uncultured         Microbacterium sp. glucosidase, Uniparc reference UPI000A2BB4A5,         Uniprot reference A0A1Y5P895.     -   Seq ID No. 889: Amino acid sequence for Periconia macrospinosa         glucosidase, Uniparc reference UPI000D5BC9BD, Uniprot reference         A0A2V1E673.     -   Seq ID No. 890: Amino acid sequence for Arthrobacter         ulcerisalmonis glucosidase, Uniparc reference UPI000F3DC30B,         Uniprot reference A0A3P5WTE2.     -   Seq ID No. 891: Amino acid sequence for Pseudarthrobacter         chlorophenolicus glucosidase, Uniparc reference UPI0001664880,         Uniprot reference B8H9A2.     -   Seq ID No. 892: Amino acid sequence for Haloplasma contractile         glucosidase, Uniparc reference UPI0002120C63, Uniprot reference         F7PUF3.     -   Seq ID No. 893: Amino acid sequence for Cochliobolus         heterostrophus glucosidase, Uniparc reference UPI0002B73341,         Uniprot reference M2ULB2.     -   Seq ID No. 894: Amino acid sequence for Microbacterium sp.         glucosidase, Uniparc reference UPI0003DE58EA, Uniprot reference         W0Z818.     -   Seq ID No. 895: Codon optimised DNA encoding 6×His         Bifidobacterium actinocoloniiforme glucosidase, Uniparc         reference UPI000503B26C, Uniprot reference A0A086YYS8.     -   Seq ID No. 896: Codon optimised DNA encoding 6×His         Bifidobacterium psychraerophilum glucosidase, Uniparc reference         UPI000502B461, Uniprot reference A0A087CD28.     -   Seq ID No. 897: Codon optimised DNA encoding 6×His Penicillium         italicum glucosidase, Uniparc reference UPI00052B8681, Uniprot         reference A0A0A2K704.     -   Seq ID No. 898: Codon optimised DNA encoding 6×His         Microbacterium trichothecenolyticum glucosidase, Uniparc         reference UPI0005ED0AE6, Uniprot reference A0A0M2HDB3.     -   Seq ID No. 899: Codon optimised DNA encoding 6×His uncultured         Clostridium sp. glucosidase, Uniparc reference UPI0008206F38,         Uniprot reference A0A1C6I6A3.     -   Seq ID No. 900: Codon optimised DNA encoding 6×His Bifiguratus         adelaidae glucosidase, Uniparc reference UPI000BC57319, Uniprot         reference A0A261Y7Q8.     -   Seq ID No. 901: Codon optimised DNA encoding 6×His Paenibacillus         thiaminolyticus glucosidase, Uniparc reference UPI000B3B73CD,         Uniprot reference A0A378ZIK3.     -   Seq ID No. 902: Codon optimised DNA encoding 6×His         Microbacterium lemovicicum glucosidase, Uniparc reference         UPI000F8FB9BA, Uniprot reference A0A3S9WE68.     -   Seq ID No. 903: Codon optimised DNA encoding 6×His Rhodococcus         erythropolis glucosidase, Uniparc reference UPI00019923DC,         Uniprot reference C1A1N4.     -   Seq ID No. 904: Codon optimised DNA encoding 6×His Cutibacterium         avidum glucosidase, Uniparc reference UPI00022C19B5, Uniprot         reference G4CZU8.     -   Seq ID No. 905: Codon optimised DNA encoding 6×His Clavibacter         michiganensis glucosidase, Uniparc reference UPI0002C5A938,         Uniprot reference M5BD10.     -   Seq ID No. 906: Codon optimised DNA encoding 6×His         Microbacterium sp. glucosidase, Uniparc reference UPI0003DE3509,         Uniprot reference W0ZC23.     -   Seq ID No. 907: Codon optimised DNA encoding 6×His         Bifidobacterium actinocoloniiforme glucosidase, Uniparc         reference UPI000507F38A, Uniprot reference A0A086YZL4.     -   Seq ID No. 908: Codon optimised DNA encoding 6×His         Bifidobacterium reuteri glucosidase, Uniparc reference         UPI000506119A, Uniprot reference A0A087CR26.     -   Seq ID No. 909: Codon optimised DNA encoding 6×His         Propionibacterium freudenreichii glucosidase, Uniparc reference         UPI0005A5CAC2, Uniprot reference A0A0A8RX48.     -   Seq ID No. 910: Codon optimised DNA encoding 6×His         Microbacterium hydrocarbonoxydans glucosidase, Uniparc reference         UPI0005EC18F9, Uniprot reference A0A0M2HRU0.     -   Seq ID No. 911: Codon optimised DNA encoding 6×His         Pseudonocardia sp. glucosidase, Uniparc reference UPI00094B6D3B,         Uniprot reference A0A1Q8KSV8.     -   Seq ID No. 912: Codon optimised DNA encoding 6×His Tuber         aestivum glucosidase, Uniparc reference UPI000BC13DF0, Uniprot         reference A0A292PKV5.     -   Seq ID No. 913: Codon optimised DNA encoding 6×His         Propionibacterium australiense glucosidase, Uniparc reference         UPI000E5B4EA5, Uniprot reference A0A383S7A9.     -   Seq ID No. 914: Codon optimised DNA encoding 6×His Fusarium sp.         glucosidase, Uniparc reference UPI001004B2C9, Uniprot reference         A0A428T6E2.     -   Seq ID No. 915: Codon optimised DNA encoding 6×His Nectria         haematococca glucosidase, Uniparc reference UPI0001B69B5C,         Uniprot reference C7ZBV0.     -   Seq ID No. 916: Codon optimised DNA encoding 6×His Actinoplanes         sp. glucosidase, Uniparc reference UPI00023EBB15, Uniprot         reference G8S3E7.     -   Seq ID No. 917: Codon optimised DNA encoding 6×His Streptomyces         fulvissimus glucosidase, Uniparc reference UPI0003289BC6,         Uniprot reference N0CMW2.     -   Seq ID No. 918: Codon optimised DNA encoding 6×His         Pestalotiopsis fici glucosidase, Uniparc reference         UPI0003E02BF1, Uniprot reference W3WXF1.     -   Seq ID No. 919: Codon optimised DNA encoding 6×His         Bifidobacterium bohemicum glucosidase, Uniparc reference         UPI0005025F2E, Uniprot reference A0A086ZGP0.     -   Seq ID No. 920: Codon optimised DNA encoding 6×His         Bifidobacterium saeculare glucosidase, Uniparc reference         UPI0005084E52, Uniprot reference A0A087D0Q2.     -   Seq ID No. 921: Codon optimised DNA encoding 6×His Bionectria         ochroleuca glucosidase, Uniparc reference UPI00059674D6, Uniprot         reference A0A0B7JW24.     -   Seq ID No. 922: Codon optimised DNA encoding 6×His         Bifidobacterium pseudocatenulatum glucosidase, Uniparc reference         UPI0006C6D6F9, Uniprot reference A0A174AU04.     -   Seq ID No. 923: Codon optimised DNA encoding 6×His         Pseudonocardia sp. glucosidase, Uniparc reference UPI00094ABAB3,         Uniprot reference A0A1Q8LPB4.     -   Seq ID No. 924: Codon optimised DNA encoding 6×His bacterium         glucosidase, Uniparc reference UPI000CB49A0B, Uniprot reference         A0A2H5Z8Q4.     -   Seq ID No. 925: Codon optimised DNA encoding 6×His Coleophoma         crateriformis glucosidase, Uniparc reference UPI000E38A995,         Uniprot reference A0A3D8Q771.     -   Seq ID No. 926: Codon optimised DNA encoding 6×His Arthrobotrys         oligospora glucosidase, Uniparc reference UPI001102DFA3, Uniprot         reference A0A4Z0Y5Y8.     -   Seq ID No. 927: Codon optimised DNA encoding 6×His Pyrenophora         teres glucosidase, Uniparc reference UPI0001ECDCBD, Uniprot         reference E3RFS2.     -   Seq ID No. 928: Codon optimised DNA encoding 6×His Gordonia         polyisoprenivorans glucosidase, Uniparc reference UPI00024F2A26,         Uniprot reference H6MTQ7.     -   Seq ID No. 929: Codon optimised DNA encoding 6×His Stigmatella         aurantiaca glucosidase, Uniparc reference UPI0000E28E5D, Uniprot         reference Q08521.     -   Seq ID No. 930: Codon optimised DNA encoding 6×His         Pestalotiopsis fici glucosidase, Uniparc reference         UPI0003E03A52, Uniprot reference W3WZ03.     -   Seq ID No. 931: Codon optimised DNA encoding 6×His         Bifidobacterium magnum glucosidase, Uniparc reference         UPI0003B7B6EE, Uniprot reference A0A087BEN9.     -   Seq ID No. 932: Codon optimised DNA encoding 6×His         Bifidobacterium stellenboschense glucosidase, Uniparc reference         UPI000503F283, Uniprot reference A0A087DFL8.     -   Seq ID No. 933: Codon optimised DNA encoding 6×His Bionectria         ochroleuca glucosidase, Uniparc reference UPI00059673EE, Uniprot         reference A0A0B7K316.     -   Seq ID No. 934: Codon optimised DNA encoding 6×His Hungatella         hathewayi glucosidase, Uniparc reference UPI0006C069F3, Uniprot         reference A0A174LVE3.     -   Seq ID No. 935: Codon optimised DNA encoding 6×His Mycetocola         reblochoni glucosidase, Uniparc reference UPI00097EB800, Uniprot         reference A0A1R4J2F9.     -   Seq ID No. 936: Codon optimised DNA encoding 6×His Nonomuraea         sp. glucosidase, Uniparc reference UPI0009ABD7B3, Uniprot         reference A0A2P9IX34.     -   Seq ID No. 937: Codon optimised DNA encoding 6×His Coleophoma         crateriformis glucosidase, Uniparc reference UPI000E391DF6,         Uniprot reference A0A3D8T9C2.     -   Seq ID No. 938: Codon optimised DNA encoding 6×His         Paenarthrobacter aurescens glucosidase, Uniparc reference         UPI0000EC83AE, Uniprot reference A1R2K1.     -   Seq ID No. 939: Codon optimised DNA encoding 6×His Kitasatospora         setae glucosidase, Uniparc reference UPI0001F21F08, Uniprot         reference E4N4F6.     -   Seq ID No. 940: Codon optimised DNA encoding 6×His Nocardiopsis         alba glucosidase, Uniparc reference UPI00027E251E, Uniprot         reference J7L3Z8.     -   Seq ID No. 941: Codon optimised DNA encoding 6×His Rhodococcus         jostii glucosidase, Uniparc reference UPI0000DBA338, Uniprot         reference Q05014.     -   Seq ID No. 942: Codon optimised DNA encoding 6×His uncultured         microorganism glucosidase, Uniparc reference UPI0003EC942D,         Uniprot reference W5X324.     -   Seq ID No. 943: Codon optimised DNA encoding 6×His         Bifidobacterium merycicum glucosidase, Uniparc reference         UPI0005084B63, Uniprot reference A0A087BJ88.     -   Seq ID No. 944: Codon optimised DNA encoding 6×His         Bifidobacterium scardovii glucosidase, Uniparc reference         UPI0005018B5B, Uniprot reference A0A087DGT3.     -   Seq ID No. 945: Codon optimised DNA encoding 6×His Fusarium         oxysporum glucosidase, Uniparc reference UPI00021EC81E, Uniprot         reference A0A0C4DJL4.     -   Seq ID No. 946: Codon optimised DNA encoding 6×His         Paraphaeosphaeria sporulosa glucosidase, Uniparc reference         UPI0007CE199C, Uniprot reference A0A177BXH1.     -   Seq ID No. 947: Codon optimised DNA encoding 6×His Clostridium         oryzae glucosidase, Uniparc reference UPI0009A50F8D, Uniprot         reference A0A1V4IY77.     -   Seq ID No. 948: Codon optimised DNA encoding 6×His Corynespora         cassiicola glucosidase, Uniparc reference UPI000D22F5EB, Uniprot         reference A0A2T2N4T6.     -   Seq ID No. 949: Codon optimised DNA encoding 6×His Choiromyces         venosus glucosidase, Uniparc reference UPI000F7332F8, Uniprot         reference A0A3N4J9R6.     -   Seq ID No. 950: Codon optimised DNA encoding 6×His         Saccharopolyspora erythraea glucosidase, Uniparc reference         UPI0000F550EC, Uniprot reference A4F982.     -   Seq ID No. 951: Codon optimised DNA encoding 6×His Streptomyces         venezuelae glucosidase, Uniparc reference UPI000204906E, Uniprot         reference F2R0Y2.     -   Seq ID No. 952: Codon optimised DNA encoding 6×His Arthrobacter         sp. glucosidase, Uniparc reference UPI00027DFDB0, Uniprot         reference J7LVB1.     -   Seq ID No. 953: Codon optimised DNA encoding 6×His         Thermobrachium celere glucosidase, Uniparc reference         UPI00033420CB, Uniprot reference R7RMQ6.     -   Seq ID No. 954: Codon optimised DNA encoding 6×His Drechslerella         stenobrocha glucosidase, Uniparc reference UPI0003EA65B9,         Uniprot reference W7HT93.     -   Seq ID No. 955: Codon optimised DNA encoding 6×His         Bifidobacterium minimum glucosidase, Uniparc reference         UPI0003B38542, Uniprot reference A0A087BMR5.     -   Seq ID No. 956: Codon optimised DNA encoding 6×His         Bifidobacterium thermacidophilum glucosidase, Uniparc reference         UPI000409825E, Uniprot reference A0A087E3K8.     -   Seq ID No. 957: Codon optimised DNA encoding 6×His Verruconis         gallopava glucosidase, Uniparc reference UPI0005C0229F, Uniprot         reference A0A0D1X9G0.     -   Seq ID No. 958: Codon optimised DNA encoding 6×His Stagonospora         sp. glucosidase, Uniparc reference UPI0007CECAD5, Uniprot         reference A0A178AER7.     -   Seq ID No. 959: Codon optimised DNA encoding 6×His Firmicutes         bacterium glucosidase, Uniparc reference UPI0009D58C49, Uniprot         reference A0A1V6ALF9.     -   Seq ID No. 960: Codon optimised DNA encoding 6×His Tuber borchii         glucosidase, Uniparc reference UPI000D50EED1, Uniprot reference         A0A2T6ZNU7.     -   Seq ID No. 961: Codon optimised DNA encoding 6×His Morchella         conica glucosidase, Uniparc reference UPI000F728167, Uniprot         reference A0A3N4KUR3.     -   Seq ID No. 962: Codon optimised DNA encoding 6×His Dictyoglomus         thermophilum glucosidase, Uniparc reference UPI00018152EB,         Uniprot reference B5YC96.     -   Seq ID No. 963: Codon optimised DNA encoding 6×His Microlunatus         phosphovorus glucosidase, Uniparc reference UPI000210C01C,         Uniprot reference F5XL24.     -   Seq ID No. 964: Codon optimised DNA encoding 6×His         Bifidobacterium asteroides glucosidase, Uniparc reference         UPI00028BB1B6, Uniprot reference K4IPD2.     -   Seq ID No. 965: Codon optimised DNA encoding 6×His Dactylellina         haptotyla glucosidase, Uniparc reference UPI00035AE576, Uniprot         reference S8BQ60.     -   Seq ID No. 966: Codon optimised DNA encoding 6×His Fusarium         oxysporum glucosidase, Uniparc reference UPI0003F2D7DD, Uniprot         reference W9JF54.     -   Seq ID No. 967: Codon optimised DNA encoding 6×His         Bifidobacterium longum glucosidase, Uniparc reference         UPI0005067FF9, Uniprot reference A0A087BRY2.     -   Seq ID No. 968: Codon optimised DNA encoding 6×His         Bifidobacterium tsurumiense glucosidase, Uniparc reference         UPI0004105550, Uniprot reference A0A087EK01.     -   Seq ID No. 969: Codon optimised DNA encoding 6×His Exophiala         spinifera glucosidase, Uniparc reference UPI0005BF9DF9, Uniprot         reference A0A0D1YG50.     -   Seq ID No. 970: Codon optimised DNA encoding 6×His Pyrenochaeta         sp. glucosidase, Uniparc reference UPI0007CE7DBA, Uniprot         reference A0A178E5I1.     -   Seq ID No. 971: Codon optimised DNA encoding 6×His Firmicutes         bacterium glucosidase, Uniparc reference UPI0009CDA3F1, Uniprot         reference A0A1V6FLK2.     -   Seq ID No. 972: Codon optimised DNA encoding 6×His Cadophora sp.         glucosidase, Uniparc reference UPI000D5A9E68, Uniprot reference         A0A2V1B599.     -   Seq ID No. 973: Codon optimised DNA encoding 6×His Morchella         conica glucosidase, Uniparc reference UPI000F7330CD, Uniprot         reference A0A3N4L4M3.     -   Seq ID No. 974: Codon optimised DNA encoding 6×His         Bifidobacterium animalis glucosidase, Uniparc reference         UPI000189C68F, Uniprot reference B8DV42.     -   Seq ID No. 975: Codon optimised DNA encoding 6×His Treponema         azotonutricium glucosidase, Uniparc reference UPI00020ED2C9,         Uniprot reference F5YGD5.     -   Seq ID No. 976: Codon optimised DNA encoding 6×His         Acidipropionibacterium acidipropionici glucosidase, Uniparc         reference UPI0002988429, Uniprot reference K7S0E5.     -   Seq ID No. 977: Codon optimised DNA encoding 6×His Salinispira         pacifica glucosidase, Uniparc reference UPI0003D93613, Uniprot         reference V5WNB6.     -   Seq ID No. 978: Codon optimised DNA encoding 6×His         Bifidobacterium mongoliense glucosidase, Uniparc reference         UPI000503588E, Uniprot reference A0A087C760.     -   Seq ID No. 979: Codon optimised DNA encoding 6×His         Bifidobacterium indicum glucosidase, Uniparc reference         UPI000499F5D1, Uniprot reference A0A087VUW4.     -   Seq ID No. 980: Codon optimised DNA encoding 6×His Brachyspira         suanatina glucosidase, Uniparc reference UPI000659257F, Uniprot         reference A0A0G4K5C2.     -   Seq ID No. 981: Codon optimised DNA encoding 6×His Phialocephala         scopiformis glucosidase, Uniparc reference UPI0007F2FD44,         Uniprot reference A0A194X8Q9.     -   Seq ID No. 982: Codon optimised DNA encoding 6×His uncultured         Microbacterium sp. glucosidase, Uniparc reference UPI000A2BB4A5,         Uniprot reference A0A1Y5P895.     -   Seq ID No. 983: Codon optimised DNA encoding 6×His Periconia         macrospinosa glucosidase, Uniparc reference UPI000D5BC9BD,         Uniprot reference A0A2V1E673.     -   Seq ID No. 984: Codon optimised DNA encoding 6×His Arthrobacter         ulcerisalmonis glucosidase, Uniparc reference UPI000F3DC30B,         Uniprot reference A0A3P5WTE2.     -   Seq ID No. 985: Codon optimised DNA encoding 6×His         Pseudarthrobacter chlorophenolicus glucosidase, Uniparc         reference UPI0001664880, Uniprot reference B8H9A2.     -   Seq ID No. 986: Codon optimised DNA encoding 6×His Haloplasma         contractile glucosidase, Uniparc reference UPI0002120C63,         Uniprot reference F7PUF3.     -   Seq ID No. 987: Codon optimised DNA encoding 6×His Cochliobolus         heterostrophus glucosidase, Uniparc reference UPI0002B73341,         Uniprot reference M2ULB2.     -   Seq ID No. 988: Codon optimised DNA encoding 6×His         Microbacterium sp. glucosidase, Uniparc reference UPI0003DE58EA,         Uniprot reference W0Z818.     -   Seq ID No. 989: Amino acid sequence for Rhodothermus marinus         rhamnosidase, Uniparc reference UPI0001A31108, Uniprot reference         D0MFR0.     -   Seq ID No. 990: Amino acid sequence for Streptomyces         bingchenggensis rhamnosidase, Uniparc reference UPI0001D90BFE,         Uniprot reference D7C463.     -   Seq ID No. 991: Amino acid sequence for Spirosoma linguale         rhamnosidase, Uniparc reference UPI0001A3AEAC, Uniprot reference         D2QL60.     -   Seq ID No. 992: Amino acid sequence for Roseburia intestinalis         rhamnosidase, Uniparc reference UPI0001CD6D48, Uniprot reference         D4L2K8.     -   Seq ID No. 993: Amino acid sequence for Draconibacterium         orientale rhamnosidase, Uniparc reference UPI000442EF22, Uniprot         reference X5DG83.     -   Seq ID No. 994: Amino acid sequence for Catenulispora acidiphila         rhamnosidase, Uniparc reference UPI00019E052B, Uniprot reference         C7QC24.     -   Seq ID No. 995: Amino acid sequence for Bacteroides         thetaiotaomicron rhamnosidase, Uniparc reference UPI000005ADE1,         Uniprot reference Q8A916.     -   Seq ID No. 996: Amino acid sequence for Opitutus terrae         rhamnosidase, Uniparc reference UPI000172B2E3, Uniprot reference         B1ZRE4.     -   Seq ID No. 997: Amino acid sequence for Lachnoclostridium         phytofermentans rhamnosidase, Uniparc reference UPI00015FE0BE,         Uniprot reference A9KJP8.     -   Seq ID No. 998: Amino acid sequence for Rhodanobacter         denitrificans rhamnosidase, Uniparc reference UPI00022DA3ED,         Uniprot reference M4NH01.     -   Seq ID No. 999: Amino acid sequence for Prevotella ruminicola         rhamnosidase, Uniparc reference UPI0001D07633, Uniprot reference         D5ETD9.     -   Seq ID No. 1000: Amino acid sequence for Aspergillus terreus         rhamnosidase, Uniparc reference UPI000259E388, Uniprot reference         I0AZ41.     -   Seq ID No. 1001: Amino acid sequence for Brachybacterium faecium         rhamnosidase, Uniparc reference UPI0001A461B6, Uniprot reference         C7MA58.     -   Seq ID No. 1002: Amino acid sequence for Flavobacterium         johnsoniae rhamnosidase, Uniparc reference UPI00006E4A0D,         Uniprot reference A5FCH3.     -   Seq ID No. 1003: Amino acid sequence for Rahnella aquatilis         rhamnosidase, Uniparc reference UPI000245C507, Uniprot reference         H2IYR2.     -   Seq ID No. 1004: Amino acid sequence for Bifidobacterium         moukalabense rhamnosidase, Uniparc reference UPI0003E4E711,         Uniprot reference W4N6H0.     -   Seq ID No. 1005: Amino acid sequence for Enterococcus         casseliflavus rhamnosidase, Uniparc reference UPI000353BD9F,         Uniprot reference S4BBS9.     -   Seq ID No. 1006: Amino acid sequence for Geobacillus sp.         rhamnosidase, Uniparc reference UPI0001789C0D, Uniprot reference         D3EED1.     -   Seq ID No. 1007: Amino acid sequence for Modestobacter marinus         rhamnosidase, Uniparc reference UPI000260A2FE, Uniprot reference         I4EYD9.     -   Seq ID No. 1008: Amino acid sequence for Pedobacter heparinus         rhamnosidase, Uniparc reference UPI00019EF0EE, Uniprot reference         C6XYM6.     -   Seq ID No. 1009: Amino acid sequence for Dyadobacter fermentans         rhamnosidase, Uniparc reference UPI00019B5915, Uniprot reference         C6VZL3.     -   Seq ID No. 1010: Amino acid sequence for Paenibacillus         mucilaginosus rhamnosidase, Uniparc reference UPI000212C156,         Uniprot reference F8FQQ3.     -   Seq ID No. 1011: Amino acid sequence for Paenibacillus sp.         rhamnosidase, Uniparc reference UPI0003E2544D, Uniprot reference         W4D866.     -   Seq ID No. 1012: Amino acid sequence for Bacteroides         thetaiotaomicron rhamnosidase, Uniparc reference UPI000005ADD2,         Uniprot reference A0A0P0FM19.     -   Seq ID No. 1013: Amino acid sequence for Chloroflexus         aurantiacus rhamnosidase, Uniparc reference UPI00005BA60B,         Uniprot reference A9WDK5.     -   Seq ID No. 1014: Amino acid sequence for Thermoclostridium         stercorarium rhamnosidase, Uniparc reference UPI00000B098C,         Uniprot reference Q9S3L0.     -   Seq ID No. 1015: Amino acid sequence for Bifidobacterium         moukalabense rhamnosidase, Uniparc reference UPI0003E5C314,         Uniprot reference W4N612.     -   Seq ID No. 1016: Amino acid sequence for Olsenella profusa         rhamnosidase, Uniparc reference UPI0003AE032C, Uniprot reference         U2USP4.     -   Seq ID No. 1017: Amino acid sequence for Kribbella flavida         rhamnosidase, Uniparc reference UPI00019BDB13, Uniprot reference         D2PMT5.     -   Seq ID No. 1018: Amino acid sequence for Caulobacter vibrioides         rhamnosidase, Uniparc reference UPI00000C7226, Uniprot reference         Q9A9K2.     -   Seq ID No. 1019: Amino acid sequence for Bacteroides         thetaiotaomicron rhamnosidase, Uniparc reference UPI000005BA09,         Uniprot reference Q8A076.     -   Seq ID No. 1020: Amino acid sequence for Rhodonellum         psychrophilum rhamnosidase, Uniparc reference UPI0003745394,         Uniprot reference U5BUY4.     -   Seq ID No. 1021: Amino acid sequence for Paenibacillus sp.         rhamnosidase, Uniparc reference UPI0004F6D660, Uniprot reference         A0A089M3T2.     -   Seq ID No. 1022: Amino acid sequence for Catenovulum agarivorans         rhamnosidase, Uniparc reference UPI0003ED860D, Uniprot reference         W7QMH5.     -   Seq ID No. 1023: Amino acid sequence for Zobellia         galactanivorans rhamnosidase, Uniparc reference UPI000217D8B1,         Uniprot reference G0L382.     -   Seq ID No. 1024: Amino acid sequence for Bacteroides         thetaiotaomicron rhamnosidase, Uniparc reference UPI000005B845,         Uniprot reference Q8A1H5.     -   Seq ID No. 1025: Amino acid sequence for Bacteroides         xylanisolvens rhamnosidase, Uniparc reference UPI0001CD02E9,         Uniprot reference D6CYE5.     -   Seq ID No. 1026: Amino acid sequence for Pseudarthrobacter         chlorophenolicus rhamnosidase, Uniparc reference UPI00018E07C6,         Uniprot reference B8HAH3.     -   Seq ID No. 1027: Amino acid sequence for Dictyoglomus         thermophilum rhamnosidase, Uniparc reference UPI0001815896,         Uniprot reference B5YC64.     -   Seq ID No. 1028: Amino acid sequence for Formosa agariphila         rhamnosidase, Uniparc reference UPI00039231C1, Uniprot reference         T2KPL4.     -   Seq ID No. 1029: Amino acid sequence for Rhodococcus jostii         rhamnosidase, Uniparc reference UPI0000DBA6EB, Uniprot reference         Q0S9T4.     -   Seq ID No. 1030: Amino acid sequence for Lactobacillus crispatus         rhamnosidase, Uniparc reference UPI0001D10896, Uniprot reference         D5GZ45.     -   Seq ID No. 1031: Amino acid sequence for Pedobacter heparinus         rhamnosidase, Uniparc reference UPI0001B17DE6, Uniprot reference         C6XVU2.     -   Seq ID No. 1032: Amino acid sequence for Spirosoma linguale         rhamnosidase, Uniparc reference UPI0001A3C989, Uniprot reference         D2QUA5.     -   Seq ID No. 1033: Amino acid sequence for Pedobacter heparinus         rhamnosidase, Uniparc reference UPI00019EE3EB, Uniprot reference         C6XU05.     -   Seq ID No. 1034: Amino acid sequence for Paenibacillus         mucilaginosus rhamnosidase, Uniparc reference UPI0003432C11,         Uniprot reference R9ULQ4.     -   Seq ID No. 1035: Amino acid sequence for Caulobacter segnis         rhamnosidase, Uniparc reference UPI0001BC0C05, Uniprot reference         D5VGD9.     -   Seq ID No. 1036: Amino acid sequence for Bacteroides         cellulosilyticus rhamnosidase, Uniparc reference UPI0001969377,         Uniprot reference E2N9B1.     -   Seq ID No. 1037: Amino acid sequence for Pedobacter heparinus         rhamnosidase, Uniparc reference UPI0001B17C60, Uniprot reference         C6Y153.     -   Seq ID No. 1038: Amino acid sequence for Formosa agariphila         rhamnosidase, Uniparc reference UPI000571C0C2, Uniprot reference         T2KNB2.     -   Seq ID No. 1039: Amino acid sequence for Lactobacillus         acidophilus rhamnosidase, Uniparc reference UPI00004C6D41,         Uniprot reference Q5FJ31.     -   Seq ID No. 1040: Amino acid sequence for Rhodopirellula baltica         rhamnosidase, Uniparc reference UPI00001AC07D, Uniprot reference         Q7UYD5.     -   Seq ID No. 1041: Amino acid sequence for Frankia inefficax         rhamnosidase, Uniparc reference UPI0001BF9A6C, Uniprot reference         E3IY10.     -   Seq ID No. 1042: Amino acid sequence for Streptomyces scabiei         rhamnosidase, Uniparc reference UPI0001B7FF91, Uniprot reference         C9Z376.     -   Seq ID No. 1043: Amino acid sequence for Flavobacterium         johnsoniae rhamnosidase, Uniparc reference UPI00006E5F74,         Uniprot reference A5FC22.     -   Seq ID No. 1044: Amino acid sequence for Streptomyces sp.         rhamnosidase, Uniparc reference UPI00034E666D, Uniprot reference         S2YWB5.     -   Seq ID No. 1045: Amino acid sequence for Acidobacterium         capsulatum rhamnosidase, Uniparc reference UPI000198DF25,         Uniprot reference C1F149.     -   Seq ID No. 1046: Amino acid sequence for Catenovulum agarivorans         rhamnosidase, Uniparc reference UPI0003ED82D3, Uniprot reference         W7QYP5.     -   Seq ID No. 1047: Amino acid sequence for Brachybacterium faecium         rhamnosidase, Uniparc reference UPI0001A461B7, Uniprot reference         C7MA59.     -   Seq ID No. 1048: Amino acid sequence for Klebsiella oxytoca         rhamnosidase, Uniparc reference UPI000243A177, Uniprot reference         A0A0J9X262.     -   Seq ID No. 1049: Amino acid sequence for Chitinophaga pinensis         rhamnosidase, Uniparc reference UPI0001B24769, Uniprot reference         C7PA70.     -   Seq ID No. 1050: Amino acid sequence for Streptomyces         bottropensis rhamnosidase, Uniparc reference UPI0002BCAF6B,         Uniprot reference M3FYL9.     -   Seq ID No. 1051: Amino acid sequence for Subdoligranulum         variabile rhamnosidase, Uniparc reference UPI0001966B28, Uniprot         reference D1PKC7.     -   Seq ID No. 1052: Amino acid sequence for Microbacterium         testaceum rhamnosidase, Uniparc reference UPI0001F8A51D, Uniprot         reference E8NDD8.     -   Seq ID No. 1053: Amino acid sequence for Solibacter usitatus         rhamnosidase, Uniparc reference UPI000053767B, Uniprot reference         Q01V09.     -   Seq ID No. 1054: Amino acid sequence for Streptosporangium         roseum rhamnosidase, Uniparc reference UPI0001A3EFE6, Uniprot         reference D2B240.     -   Seq ID No. 1055: Amino acid sequence for alpha proteobacterium         rhamnosidase, Uniparc reference UPI0006CE1E82, Uniprot reference         A0A0N1BME3.     -   Seq ID No. 1056: Amino acid sequence for Solitalea canadensis         rhamnosidase, Uniparc reference UPI000247229E, Uniprot reference         H8KPI7.     -   Seq ID No. 1057: Amino acid sequence for Parabacteroides         goldsteinii rhamnosidase, Uniparc reference UPI0002CB9583,         Uniprot reference S0GSF0.     -   Seq ID No. 1058: Amino acid sequence for Cyclobacterium marinum         rhamnosidase, Uniparc reference UPI00021B9B33, Uniprot reference         G0J630.     -   Seq ID No. 1059: Amino acid sequence for Solibacter usitatus         rhamnosidase, Uniparc reference UPI0000533669, Uniprot reference         Q01TX2.     -   Seq ID No. 1060: Amino acid sequence for Lunatimonas lonarensis         rhamnosidase, Uniparc reference UPI00032D8F6D, Uniprot reference         R7ZW70.     -   Seq ID No. 1061: Amino acid sequence for Rhizobium leguminosarum         rhamnosidase, Uniparc reference UPI0000D713F2, Uniprot reference         Q1M7P3.     -   Seq ID No. 1062: Amino acid sequence for Streptosporangium         roseum rhamnosidase, Uniparc reference UPI0001A3EBEB, Uniprot         reference D2AYU9.     -   Seq ID No. 1063: Amino acid sequence for Parabacteroides         distasonis rhamnosidase, Uniparc reference UPI000156F115,         Uniprot reference A6LBL4.     -   Seq ID No. 1064: Amino acid sequence for Lachnospiraceae         bacterium rhamnosidase, Uniparc reference UPI0003375A10, Uniprot         reference R9K6L6.     -   Seq ID No. 1065: Amino acid sequence for Chitinophaga pinensis         rhamnosidase, Uniparc reference UPI0001A2F0FA, Uniprot reference         C7P9Y8.     -   Seq ID No. 1066: Amino acid sequence for Caulobacter segnis         rhamnosidase, Uniparc reference UPI0001B00015, Uniprot reference         D5VGC3.     -   Seq ID No. 1067: Amino acid sequence for Pedobacter heparinus         rhamnosidase, Uniparc reference UPI00019EF6E1, Uniprot reference         C6Y145.     -   Seq ID No. 1068: Amino acid sequence for Pedobacter heparinus         rhamnosidase, Uniparc reference UPI00019EE1A9, Uniprot reference         C6Y2X3.     -   Seq ID No. 1069: Amino acid sequence for Deltaproteobacteria         bacterium rhamnosidase, Uniparc reference UPI000C8D4928, Uniprot         reference A0A2D5SK32.     -   Seq ID No. 1070: Amino acid sequence for Thermobaculum terrenum         rhamnosidase, Uniparc reference UPI00019BFDCE, Uniprot reference         D1CHL4.     -   Seq ID No. 1071: Amino acid sequence for Opitutus terrae         rhamnosidase, Uniparc reference UPI000172B62A, Uniprot reference         B1ZY35.     -   Seq ID No. 1072: Amino acid sequence for Kribbella flavida         rhamnosidase, Uniparc reference UPI00019BFABB, Uniprot reference         D2PXQ4.     -   Seq ID No. 1073: Amino acid sequence for Streptomyces scabiei         rhamnosidase, Uniparc reference UPI0001B80091, Uniprot reference         C9Z391.     -   Seq ID No. 1074: Amino acid sequence for Actinoplanes sp.         rhamnosidase, Uniparc reference UPI00023EC5D1, Uniprot reference         G8S540.     -   Seq ID No. 1075: Amino acid sequence for Asticcacaulis sp.         rhamnosidase, Uniparc reference UPI0003C3CD2B, Uniprot reference         V4NSJ1.     -   Seq ID No. 1076: Amino acid sequence for Kribbella flavida         rhamnosidase, Uniparc reference UPI00019BF65D, Uniprot reference         D2PT74.     -   Seq ID No. 1077: Amino acid sequence for Bacillus sp.         rhamnosidase, Uniparc reference UPI00000BC760, Uniprot reference         Q93RE7.     -   Seq ID No. 1078: Amino acid sequence for Flavobacterium         johnsoniae rhamnosidase, Uniparc reference UPI00006E5FAB,         Uniprot reference A5FCG3.     -   Seq ID No. 1079: Amino acid sequence for Lunatimonas lonarensis         rhamnosidase, Uniparc reference UPI00032EEB9C, Uniprot reference         R7ZS84.     -   Seq ID No. 1080: Amino acid sequence for Eisenbergiella         massiliensis rhamnosidase, Uniparc reference UPI0004B2D794,         Uniprot reference A0A3E3IGR6.     -   Seq ID No. 1081: Amino acid sequence for Catenovulum agarivorans         rhamnosidase, Uniparc reference UPI0003ED7515, Uniprot reference         W7QF25.     -   Seq ID No. 1083: Codon optimised DNA encoding 6×His Rhodothermus         marinus rhamnosidase, Uniparc reference UPI0001A31108, Uniprot         reference D0MFR0.     -   Seq ID No. 1084: Codon optimised DNA encoding 6×His Streptomyces         bingchenggensis rhamnosidase, Uniparc reference UPI0001D90BFE,         Uniprot reference D7C463.     -   Seq ID No. 1085: Codon optimised DNA encoding 6×His Spirosoma         linguale rhamnosidase, Uniparc reference UPI0001A3AEAC, Uniprot         reference D2QL60.     -   Seq ID No. 1086: Codon optimised DNA encoding 6×His Roseburia         intestinalis rhamnosidase, Uniparc reference UPI0001CD6D48,         Uniprot reference D4L2K8.     -   Seq ID No. 1087: Codon optimised DNA encoding 6×His         Draconibacterium orientale rhamnosidase, Uniparc reference         UPI000442EF22, Uniprot reference X5DG83.     -   Seq ID No. 1088: Codon optimised DNA encoding 6×His         Catenulispora acidiphila rhamnosidase, Uniparc reference         UPI00019E052B, Uniprot reference C7QC24.     -   Seq ID No. 1089: Codon optimised DNA encoding 6×His Bacteroides         thetaiotaomicron rhamnosidase, Uniparc reference UPI000005ADE1,         Uniprot reference Q8A916.     -   Seq ID No. 1090: Codon optimised DNA encoding 6×His Opitutus         terrae rhamnosidase, Uniparc reference UPI000172B2E3, Uniprot         reference B1ZRE4.     -   Seq ID No. 1091: Codon optimised DNA encoding 6×His         Lachnoclostridium phytofermentans rhamnosidase, Uniparc         reference UPI00015FE0BE, Uniprot reference A9KJ P8.     -   Seq ID No. 1092: Codon optimised DNA encoding 6×His         Rhodanobacter denitrificans rhamnosidase, Uniparc reference         UPI00022DA3ED, Uniprot reference M4NH01.     -   Seq ID No. 1093: Codon optimised DNA encoding 6×His Prevotella         ruminicola rhamnosidase, Uniparc reference UPI0001D07633,         Uniprot reference D5ETD9.     -   Seq ID No. 1094: Codon optimised DNA encoding 6×His Aspergillus         terreus rhamnosidase, Uniparc reference UPI000259E388, Uniprot         reference I0AZ41.     -   Seq ID No. 1095: Codon optimised DNA encoding 6×His         Brachybacterium faecium rhamnosidase, Uniparc reference         UPI0001A461B6, Uniprot reference C7MA58.     -   Seq ID No. 1096: Codon optimised DNA encoding 6×His         Flavobacterium johnsoniae rhamnosidase, Uniparc reference         UPI00006E4A0D, Uniprot reference A5FCH3.     -   Seq ID No. 1097: Codon optimised DNA encoding 6×His Rahnella         aquatilis rhamnosidase, Uniparc reference UPI000245C507, Uniprot         reference H2IYR2.     -   Seq ID No. 1098: Codon optimised DNA encoding 6×His         Bifidobacterium moukalabense rhamnosidase, Uniparc reference         UPI0003E4E711, Uniprot reference W4N6H0.     -   Seq ID No. 1099: Codon optimised DNA encoding 6×His Enterococcus         casseliflavus rhamnosidase, Uniparc reference UPI000353BD9F,         Uniprot reference S4BBS9.     -   Seq ID No. 1100: Codon optimised DNA encoding 6×His Geobacillus         sp. rhamnosidase, Uniparc reference UPI0001789C0D, Uniprot         reference D3EED1.     -   Seq ID No. 1101: Codon optimised DNA encoding 6×His         Modestobacter marinus rhamnosidase, Uniparc reference         UPI000260A2FE, Uniprot reference I4EYD9.     -   Seq ID No. 1102: Codon optimised DNA encoding 6×His Pedobacter         heparinus rhamnosidase, Uniparc reference UPI00019EF0EE, Uniprot         reference C6XYM6.     -   Seq ID No. 1103: Codon optimised DNA encoding 6×His Dyadobacter         fermentans rhamnosidase, Uniparc reference UPI00019B5915,         Uniprot reference C6VZL3.     -   Seq ID No. 1104: Codon optimised DNA encoding 6×His         Paenibacillus mucilaginosus rhamnosidase, Uniparc reference         UPI000212C156, Uniprot reference F8FQQ3.     -   Seq ID No. 1105: Codon optimised DNA encoding 6×His         Paenibacillus sp. rhamnosidase, Uniparc reference UPI0003E2544D,         Uniprot reference W4D866.     -   Seq ID No. 1106: Codon optimised DNA encoding 6×His Bacteroides         thetaiotaomicron rhamnosidase, Uniparc reference UPI000005ADD2,         Uniprot reference A0A0P0FM19.     -   Seq ID No. 1107: Codon optimised DNA encoding 6×His Chloroflexus         aurantiacus rhamnosidase, Uniparc reference UPI00005BA60B,         Uniprot reference A9WDK5.     -   Seq ID No. 1108: Codon optimised DNA encoding 6×His         Thermoclostridium stercorarium rhamnosidase, Uniparc reference         UPI00000B098C, Uniprot reference Q9S3L0.     -   Seq ID No. 1109: Codon optimised DNA encoding 6×His         Bifidobacterium moukalabense rhamnosidase, Uniparc reference         UPI0003E5C314, Uniprot reference W4N6I2.     -   Seq ID No. 1110: Codon optimised DNA encoding 6×His Olsenella         profusa rhamnosidase, Uniparc reference UPI0003AE032C, Uniprot         reference U2USP4.     -   Seq ID No. 1111: Codon optimised DNA encoding 6×His Kribbella         flavida rhamnosidase, Uniparc reference UPI00019BDB13, Uniprot         reference D2PMT5.     -   Seq ID No. 1112: Codon optimised DNA encoding 6×His Caulobacter         vibrioides rhamnosidase, Uniparc reference UPI00000C7226,         Uniprot reference Q9A9K2.     -   Seq ID No. 1113: Codon optimised DNA encoding 6×His Bacteroides         thetaiotaomicron rhamnosidase, Uniparc reference UPI000005BA09,         Uniprot reference Q8A076.     -   Seq ID No. 1114: Codon optimised DNA encoding 6×His Rhodonellum         psychrophilum rhamnosidase, Uniparc reference UPI0003745394,         Uniprot reference U5BUY4.     -   Seq ID No. 1115: Codon optimised DNA encoding 6×His         Paenibacillus sp. rhamnosidase, Uniparc reference UPI0004F6D660,         Uniprot reference A0A089M3T2.     -   Seq ID No. 1116: Codon optimised DNA encoding 6×His Catenovulum         agarivorans rhamnosidase, Uniparc reference UPI0003ED860D,         Uniprot reference W7QMH5.     -   Seq ID No. 1117: Codon optimised DNA encoding 6×His Zobellia         galactanivorans rhamnosidase, Uniparc reference UPI000217D8B1,         Uniprot reference G0L382.     -   Seq ID No. 1118: Codon optimised DNA encoding 6×His Bacteroides         thetaiotaomicron rhamnosidase, Uniparc reference UPI000005B845,         Uniprot reference Q8A1H5.     -   Seq ID No. 1119: Codon optimised DNA encoding 6×His Bacteroides         xylanisolvens rhamnosidase, Uniparc reference UPI0001CD02E9,         Uniprot reference D6CYE5.     -   Seq ID No. 1120: Codon optimised DNA encoding 6×His         Pseudarthrobacter chlorophenolicus rhamnosidase, Uniparc         reference UPI00018E07C6, Uniprot reference B8HAH3.     -   Seq ID No. 1121: Codon optimised DNA encoding 6×His Dictyoglomus         thermophilum rhamnosidase, Uniparc reference UPI0001815896,         Uniprot reference B5YC64.     -   Seq ID No. 1122: Codon optimised DNA encoding 6×His Formosa         agariphila rhamnosidase, Uniparc reference UPI00039231C1,         Uniprot reference T2KPL4.     -   Seq ID No. 1123: Codon optimised DNA encoding 6×His Rhodococcus         jostii rhamnosidase, Uniparc reference UPI0000DBA6EB, Uniprot         reference Q0S9T4.     -   Seq ID No. 1124: Codon optimised DNA encoding 6×His         Lactobacillus crispatus rhamnosidase, Uniparc reference         UPI0001D10896, Uniprot reference D5GZ45.     -   Seq ID No. 1125: Codon optimised DNA encoding 6×His Pedobacter         heparinus rhamnosidase, Uniparc reference UPI0001B17DE6, Uniprot         reference C6XVU2.     -   Seq ID No. 1126: Codon optimised DNA encoding 6×His Spirosoma         linguale rhamnosidase, Uniparc reference UPI0001A3C989, Uniprot         reference D2QUA5.     -   Seq ID No. 1127: Codon optimised DNA encoding 6×His Pedobacter         heparinus rhamnosidase, Uniparc reference UPI00019EE3EB, Uniprot         reference C6XU05.     -   Seq ID No. 1128: Codon optimised DNA encoding 6×His         Paenibacillus mucilaginosus rhamnosidase, Uniparc reference         UPI0003432C11, Uniprot reference R9ULQ4.     -   Seq ID No. 1129: Codon optimised DNA encoding 6×His Caulobacter         segnis rhamnosidase, Uniparc reference UPI0001BC0C05, Uniprot         reference D5VGD9.     -   Seq ID No. 1130: Codon optimised DNA encoding 6×His Bacteroides         cellulosilyticus rhamnosidase, Uniparc reference UPI0001969377,         Uniprot reference E2N9B1.     -   Seq ID No. 1131: Codon optimised DNA encoding 6×His Pedobacter         heparinus rhamnosidase, Uniparc reference UPI0001B17C60, Uniprot         reference C6Y153.     -   Seq ID No. 1132: Codon optimised DNA encoding 6×His Formosa         agariphila rhamnosidase, Uniparc reference UPI000571C0C2,         Uniprot reference T2KNB2.     -   Seq ID No. 1133: Codon optimised DNA encoding 6×His         Lactobacillus acidophilus rhamnosidase, Uniparc reference         UPI00004C6D41, Uniprot reference Q5FJ31.     -   Seq ID No. 1134: Codon optimised DNA encoding 6×His         Rhodopirellula baltica rhamnosidase, Uniparc reference         UPI00001AC07D, Uniprot reference Q7UYD5.     -   Seq ID No. 1135: Codon optimised DNA encoding 6×His Frankia         inefficax rhamnosidase, Uniparc reference UPI0001BF9A6C, Uniprot         reference E3IY10.     -   Seq ID No. 1136: Codon optimised DNA encoding 6×His Streptomyces         scabiei rhamnosidase, Uniparc reference UPI0001B7FF91, Uniprot         reference C9Z376.     -   Seq ID No. 1137: Codon optimised DNA encoding 6×His         Flavobacterium johnsoniae rhamnosidase, Uniparc reference         UPI00006E5F74, Uniprot reference A5FC22.     -   Seq ID No. 1138: Codon optimised DNA encoding 6×His Streptomyces         sp. rhamnosidase, Uniparc reference UPI00034E666D, Uniprot         reference S2YWB5.     -   Seq ID No. 1139: Codon optimised DNA encoding 6×His         Acidobacterium capsulatum rhamnosidase, Uniparc reference         UPI000198DF25, Uniprot reference C1F149.     -   Seq ID No. 1140: Codon optimised DNA encoding 6×His Catenovulum         agarivorans rhamnosidase, Uniparc reference UPI0003ED82D3,         Uniprot reference W7QYP5.     -   Seq ID No. 1141: Codon optimised DNA encoding 6×His         Brachybacterium faecium rhamnosidase, Uniparc reference         UPI0001A461B7, Uniprot reference C7MA59.     -   Seq ID No. 1142: Codon optimised DNA encoding 6×His Klebsiella         oxytoca rhamnosidase, Uniparc reference UPI000243A177, Uniprot         reference A0A0J9X262.     -   Seq ID No. 1143: Codon optimised DNA encoding 6×His Chitinophaga         pinensis rhamnosidase, Uniparc reference UPI0001B24769, Uniprot         reference C7PA70.     -   Seq ID No. 1144: Codon optimised DNA encoding 6×His Streptomyces         bottropensis rhamnosidase, Uniparc reference UPI0002BCAF6B,         Uniprot reference M3FYL9.     -   Seq ID No. 1145: Codon optimised DNA encoding 6×His         Subdoligranulum variabile rhamnosidase, Uniparc reference         UPI0001966B28, Uniprot reference D1PKC7.     -   Seq ID No. 1146: Codon optimised DNA encoding 6×His         Microbacterium testaceum rhamnosidase, Uniparc reference         UPI0001F8A51D, Uniprot reference E8NDD8.     -   Seq ID No. 1147: Codon optimised DNA encoding 6×His Solibacter         usitatus rhamnosidase, Uniparc reference UPI000053767B, Uniprot         reference Q01V09.     -   Seq ID No. 1148: Codon optimised DNA encoding 6×His         Streptosporangium roseum rhamnosidase, Uniparc reference         UPI0001A3EFE6, Uniprot reference D2B240.     -   Seq ID No. 1149: Codon optimised DNA encoding 6×His alpha         proteobacterium rhamnosidase, Uniparc reference UPI0006CE1E82,         Uniprot reference A0A0N1BME3.     -   Seq ID No. 1150: Codon optimised DNA encoding 6×His Solitalea         canadensis rhamnosidase, Uniparc reference UPI000247229E,         Uniprot reference H8KPI7.     -   Seq ID No. 1151: Codon optimised DNA encoding 6×His         Parabacteroides goldsteinii rhamnosidase, Uniparc reference         UPI0002CB9583, Uniprot reference S0GSF0.     -   Seq ID No. 1152: Codon optimised DNA encoding 6×His         Cyclobacterium marinum rhamnosidase, Uniparc reference         UPI00021B9B33, Uniprot reference G0J630.     -   Seq ID No. 1153: Codon optimised DNA encoding 6×His Solibacter         usitatus rhamnosidase, Uniparc reference UPI0000533669, Uniprot         reference Q01TX2.     -   Seq ID No. 1154: Codon optimised DNA encoding 6×His Lunatimonas         lonarensis rhamnosidase, Uniparc reference UPI00032D8F6D,         Uniprot reference R7ZW70.     -   Seq ID No. 1155: Codon optimised DNA encoding 6×His Rhizobium         leguminosarum rhamnosidase, Uniparc reference UPI0000D713F2,         Uniprot reference Q1M7P3.     -   Seq ID No. 1156: Codon optimised DNA encoding 6×His         Streptosporangium roseum rhamnosidase, Uniparc reference         UPI0001A3EBEB, Uniprot reference D2AYU9.     -   Seq ID No. 1157: Codon optimised DNA encoding 6×His         Parabacteroides distasonis rhamnosidase, Uniparc reference         UPI000156F115, Uniprot reference A6LBL4.     -   Seq ID No. 1158: Codon optimised DNA encoding 6×His         Lachnospiraceae bacterium rhamnosidase, Uniparc reference         UPI0003375A10, Uniprot reference R9K6L6.     -   Seq ID No. 1159: Codon optimised DNA encoding 6×His Chitinophaga         pinensis rhamnosidase, Uniparc reference UPI0001A2F0FA, Uniprot         reference C7P9Y8.     -   Seq ID No. 1160: Codon optimised DNA encoding 6×His Caulobacter         segnis rhamnosidase, Uniparc reference UPI0001BC0C15, Uniprot         reference D5VGC3.     -   Seq ID No. 1161: Codon optimised DNA encoding 6×His Pedobacter         heparinus rhamnosidase, Uniparc reference UPI00019EF6E1, Uniprot         reference C6Y145.     -   Seq ID No. 1162: Codon optimised DNA encoding 6×His Pedobacter         heparinus rhamnosidase, Uniparc reference UPI00019EE1A9, Uniprot         reference C6Y2X3.     -   Seq ID No. 1163: Codon optimised DNA encoding 6×His         Deltaproteobacteria bacterium rhamnosidase, Uniparc reference         UPI000C8D4928, Uniprot reference A0A2D5SK32.     -   Seq ID No. 1164: Codon optimised DNA encoding 6×His         Thermobaculum terrenum rhamnosidase, Uniparc reference         UPI00019BFDCE, Uniprot reference D1CHL4.     -   Seq ID No. 1165: Codon optimised DNA encoding 6×His Opitutus         terrae rhamnosidase, Uniparc reference UPI000172B62A, Uniprot         reference B1ZY35.     -   Seq ID No. 1166: Codon optimised DNA encoding 6×His Kribbella         flavida rhamnosidase, Uniparc reference UPI00019BFABB, Uniprot         reference D2PXQ4.     -   Seq ID No. 1167: Codon optimised DNA encoding 6×His Streptomyces         scabiei rhamnosidase, Uniparc reference UPI0001B80091, Uniprot         reference C9Z391.     -   Seq ID No. 1168: Codon optimised DNA encoding 6×His Actinoplanes         sp. rhamnosidase, Uniparc reference UPI00023EC5D1, Uniprot         reference G8S540.     -   Seq ID No. 1169: Codon optimised DNA encoding 6×His         Asticcacaulis sp. rhamnosidase, Uniparc reference UPI0003C3CD2B,         Uniprot reference V4NSJ1.     -   Seq ID No. 1170: Codon optimised DNA encoding 6×His Kribbella         flavida rhamnosidase, Uniparc reference UPI00019BF65D, Uniprot         reference D2PT74.     -   Seq ID No. 1171: Codon optimised DNA encoding 6×His Bacillus sp.         rhamnosidase, Uniparc reference UPI00000BC760, Uniprot reference         Q93RE7.     -   Seq ID No. 1172: Codon optimised DNA encoding 6×His         Flavobacterium johnsoniae rhamnosidase, Uniparc reference         UPI00006E5FAB, Uniprot reference A5FCG3.     -   Seq ID No. 1173: Codon optimised DNA encoding 6×His Lunatimonas         lonarensis rhamnosidase, Uniparc reference UPI00032EEB9C,         Uniprot reference R7ZS84.     -   Seq ID No. 1174: Codon optimised DNA encoding 6×His         Eisenbergiella massiliensis rhamnosidase, Uniparc reference         UPI0004B2D794, Uniprot reference A0A3E3IGR6.     -   Seq ID No. 1175: Codon optimised DNA encoding 6×His Catenovulum         agarivorans rhamnosidase, Uniparc reference UPI0003ED7515,         Uniprot reference W7QF25.     -   Seq ID No. 1176: Codon optimised DNA encoding 6×His Streptomyces         avermitilis rhamnosidase, Uniparc reference UPI0000184198,         Uniprot reference Q82PP4.     -   Seq ID No. 1177: N-terminal His-tag/linker     -   Seq ID No. 1178: C-terminal linker/His tag     -   Seq ID No. 1179: Amino acid sequence for Variant G1.     -   Seq ID No. 1180: Amino acid sequence for Variant G2.     -   Seq ID No. 1181: Amino acid sequence for Variant G3.     -   Seq ID No. 1182: Amino acid sequence for Variant G4.     -   Seq ID No. 1183: Amino acid sequence for Variant G5.     -   Seq ID No. 1184: Codon optimised DNA encoding N-terminal 6×His         Variant G1.     -   Seq ID No. 1185: Codon optimised DNA encoding N-terminal 6×His         Variant G2.     -   Seq ID No. 1186: Codon optimised DNA encoding N-terminal 6×His         Variant G3.     -   Seq ID No. 1187: Codon optimised DNA encoding N-terminal 6×His         Variant G4.     -   Seq ID No. 1188: Codon optimised DNA encoding N-terminal 6×His         Variant G5.     -   Seq ID No. 1189: Amino acid sequence for Variant R1.     -   Seq ID No. 1190: Amino acid sequence for Variant R2.     -   Seq ID No. 1191: Amino acid sequence for Variant R3.     -   Seq ID No. 1192: Amino acid sequence for Variant R4.     -   Seq ID No. 1193: Amino acid sequence for Variant R5.     -   Seq ID No. 1194: Codon optimised DNA encoding C-terminal 6×His         Variant R1.     -   Seq ID No. 1195: Codon optimised DNA encoding C-terminal 6×His         Variant R2.     -   Seq ID No. 1196: Codon optimised DNA encoding C-terminal 6×His         Variant R3.     -   Seq ID No. 1197: Codon optimised DNA encoding C-terminal 6×His         Variant R4.     -   Seq ID No. 1198: Codon optimised DNA encoding C-terminal 6×His         Variant R5.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned previously, saponins are steroid or terpenoid glycosides which have a broad range of uses. Current approaches to obtaining certain saponins in suitable quantities and of suitable purities are limiting. The present inventors have surprisingly found that enzymatic modification of saponins can facilitate improved availability of saponins of interest and/or facilitate removal of undesired saponin components. The present invention therefore provides methods for the enzymatic modification of saponins, products made by such methods, uses of said products and associated aspects. In methods of the invention a starting saponin (i.e. a saponin to be modified by an enzyme) is converted into a product saponin (i.e. the saponin resulting from enzymatic modification of the starting saponin).

Engineered glucosidase polypeptides of the present invention may be used in methods for the enzymatic modification of saponins.

Engineered rhamnosidase polypeptides of the present invention may be used in methods for the enzymatic modification of saponins.

Objectives

The present invention can be applied to achieve a plurality of objectives, such as: (i) improving the yield of saponins of interest obtainable from a given starting material; (ii) broadening the range of starting materials suitable for obtaining saponins of interest; and/or (iii) convenient removal of undesired saponins from saponins of interest.

Where supply is constrained for a starting material from which a saponin of interest is isolated, achieving the maximum yield of the saponin of interest is clearly important. Independent of the efficiency of extraction and separation processes which would generally be adapted for optimal isolation of existing saponin of interest, the present invention may be applied to increase the amount of a saponin of interest which may be obtained from a given starting material. Enzymatic modification of other saponins present in the starting material to form a saponin of interest can increase the amount of the saponin of interest which may be obtained.

Saponins may be obtained from a broad range of starting materials. The presence of specific saponins and their levels in plant material may depend on a range of factors such as a plant species, tissue, age, season, environmental conditions and the like. Variation may be observed between plants (such as trees) of the same species (see, for example, WO2018057031). The burden associated with extraction and/or isolation of a saponin of interest may mean that certain potential sources of the saponin of interest are not commercially viable, due to the saponin of interest being present at relatively low levels. Enzymatic modification of other saponins present in the starting material to form a saponin of interest can expand the range of viable starting materials for obtaining the saponin of interest.

It is well understood that different saponins may have different activity profiles—both positive/desired activities and negative/undesired activities. Some uses of saponins require a high degree of purification and separating a saponin of interest from other saponins, particularly those of similar structure or physical properties, can be burdensome. Enzymatic modification of such other saponins may alter their physical properties and may thereby facilitate separation from a saponin of interest. Other uses of saponins may not require a high degree of purity per se, nevertheless it may still be desirable to remove or reduce the amount of a particular saponin component (or components) within a saponin mixture without burdensome chromatographic methods. Enzymatic modification can facilitate removal or reduction in the level of a particular saponin component within a saponin mixture without the need for chromatographic means.

Saponins

The methods of the present invention require a starting saponin (i.e. a saponin which is intended to be enzymatically modified). The starting saponin may be a naturally occurring saponin (i.e. a steroid or terpenoid glycoside found in nature) or an artificially created saponin (i.e. a steroid or terpenoid glycoside not found in nature).

In some embodiments the starting saponin is a steroid glycoside, in other embodiments the starting saponin is a terpenoid glycoside, especially a triterpenoid glycoside.

Naturally occurring starting saponins may be obtained by extraction or may be prepared synthetically (fully or semi-synthetically).

Naturally occurring starting saponins include those obtainable from, such as obtained from, plants of the genera Gypsophilia, Saponaria or Quillaja (Bomford, 1992). Especially of interest are starting saponins obtainable from plants of Quillaja species. Particular starting saponins of interest include those obtainable from Quillaja brasiliensis or Quillaja saponaria. In one embodiment the starting saponin is obtainable from Quillaja saponaria, such as obtained from Quillaja saponaria. In one embodiment the starting saponin is obtainable from Quillaja brasiliensis, such as obtained from Quillaja brasiliensis.

In certain embodiments the starting saponin is a quillaic acid glycoside. In certain embodiments the starting saponin is a phytolaccinic acid glycoside. In certain embodiments the starting saponin is an echinocystic acid glycoside. In certain embodiments the starting saponin is a 22-beta-hydroxylated quillaic acid glycoside. In certain embodiments the starting saponin is an gypsogenin glycoside.

Analysis of water/methanol extracts of Quillaja saponaria bark by liquid chromatography/mass spectrometry has revealed over 100 saponins (Nyberg, 2000; Nyberg, 2003; Kite, 2004). Quillaja brasiliensis extracts have also been described, with many saponin components in Quillaja brasiliensis extracts corresponding to saponins found in Quillaja saponaria extracts. (Wallace, 2017; Wallace, 2019).

The following text describes particular quillaic acid derived starting and product saponins which are grouped by ‘family’. Each family has one or more common structural features which characterise the family relative to other families. Individual components within each family also display certain structural features which characterise the component relative to other components of the family, including: xylose or rhamnose chemotype—the presence of a xylose or rhamnose residue in the C3 saccharide; A or B isomers—A having the acyl chain linked through the 4-position of the D-fucose, B at having the acyl chain linked through the 3-position of the D-fucose; V1 and V2—the presence of a terminal apiose or xylose residue in the C28 saccharide (in other components of a family this terminal residue may also be absent). The text focuses on components which typically have a significant presence in Quillaja saponaria aqueous extracts, but it will be appreciated that (i) other components of a family also exist and (ii) the proportions of different components of a family may vary both between families and between different saponin sources (Kite, 2004). The specific extraction method used may also influence the proportions of different components obtained.

A and B isomers may be separable using chromatographic techniques. However, under suitable solvent conditions these isomers will revert to equilibrium proportions (see e.g. Cleland, 1996). Xylose and rhamnose chemotypes typically elute closely, depending on chromatographic technique the rhamnose chemotype may form a minor peak closely preceding or overlapping with the main peak for the family.

Those skilled in the art will also recognise that the structures described contain ionisable groups and under appropriate circumstances may exist in dissociated forms or as salts. Structures are generally shown with the glucuronate moiety in ionised form and the indicated molecular weight is calculated directly from the ion shown (corresponding to the monoisotopic m/z observed with negative mode electrospray mass spectrometry), however, all non-dissociated, dissociated and salt forms are intended to be encompassed by the recited definitions. Salts are desirably pharmaceutically acceptable, although non-pharmaceutically acceptable salts can nevertheless be useful during manufacture of pharmaceuticals or for non-pharmaceutical uses.

Starting saponins obtainable from Quillaja saponaria include:

-   -   QS-18 family components (i.e. triterpenoid glycosides having         beta-O-glucopyranosylation at the C3 position of the L-rhamnose         moiety of QS-21 family components), such as:         -   ‘QS-18 2150 A component’, being the triterpenoid glycosides             identified as part of the QS-18 main peak in FIG. 2 and             having a m/z of 2150 with negative mode electrospray mass             spectrometry. The QS-18 2150 A component is believed to be             identified in Kite 2004 as Peak 76 and corresponds to the             A-isomer xylose chemotype structures B4 (apiose isomer) and             B6 (xylose isomer) characterised in Nyberg 2000 and             Nyberg 2003. The QS-18 2150 A component may consist of QS-18             2150 A V1 (i.e. apiose isomer):

-   -   and/or QS-18 2150 A V2 (i.e. xylose isomer):

-   -   -   ‘QS-18 2018 A component’, being the triterpenoid glycosides             identified as part of the QS-18 main peak in FIG. 2 and             having m/z of 2018 with negative mode electrospray mass             spectrometry. The QS-18 A 2018 component is believed to be             identified in Kite 2004 as Peak 73 and corresponds to the             A-isomer xylose chemotype structure B2 characterised in             Nyberg 2000 and Nyberg 2003. The QS-18 2018 A component may             consist of QS-18 2018 A:

-   -   -   ‘QS-18 2164 A component’, being the triterpenoid glycosides             identified as part of the QS-18 main peak in FIG. 2 and             having a m/z of 2164 with negative mode electrospray mass             spectrometry. The QS-18 2164 A component is believed to be             identified in Kite 2004 as Peak 74 and corresponds to the             A-isomer rhamnose chemotype structures B3 (apiose isomer)             and B5 (xylose isomer) characterised in Nyberg 2000 and             Nyberg 2003. The QS-18 2164 A component may consist of QS-18             2164 A V1 (i.e. apiose isomer):

-   -   and/or QS-18 2164 A V2 (i.e. xylose isomer):

-   -   -   ‘QS-18 2150 B component’, being triterpenoid glycosides             having a m/z of 2150 with negative mode electrospray mass             spectrometry. The QS-18 2150 B component corresponds to the             B-isomer xylose chemotype structures B4a (apiose isomer) and             B6a (xylose isomer) characterised in Nyberg 2000 and             Nyberg 2003. The QS-18 2150 B component may consist of QS-18             2150 B V1 (i.e. apiose isomer):

-   -   and/or QS-18 2150 B V2 (i.e. xylose isomer):

-   -   -   ‘QS-18 2018 B component’, being triterpenoid glycosides             having a m/z of 2018 with negative mode electrospray mass             spectrometry. The QS-18 2018 B component corresponds to the             B-isomer xylose chemotype structure B2a characterised in             Nyberg 2000 and Nyberg 2003. The QS-18 2018 B component may             consist of QS-18 2018 B:

-   -   -   ‘QS-18 2164 B component’, being triterpenoid glycosides             having a m/z of 2164 with negative mode electrospray mass             spectrometry. The QS-18 2164 B corresponds to the B-isomer             rhamnose chemotype structures B3a (apiose isomer) and B5a             (xylose isomer). The QS-18 2164 B component may consist of             QS-18 2164 B V1 (i.e. apiose isomer):

-   -   and/or QS-18 2164 B V2 (i.e. xylose isomer):

-   -   desglucosyl-QS-17 family components (i.e. triterpenoid         glycosides having alpha-O-rhamnosylation at the C2 position of         the arabinofuranose moiety of QS-21 family components but         lacking the glycosylation of QS-17 family components), such as:         -   ‘desglucosyl-QS-17 2134 A component’, being triterpenoid             glycosides having a m/z of 2134 with negative mode             electrospray mass spectrometry. The desglucosyl-QS-17 2134 A             component is believed to be identified in Kite 2004 as Peak             75 and corresponds to A-isomers of the xylose chemotype. The             desglucosyl-QS-17 2134 A component may consist of             desglucosyl-QS-17 2134 A V1 (i.e. apiose isomer):

-   -   and/or desglucosyl-QS-17 2134 A V2 (i.e. xylose isomer):

-   -   -   ‘desglucosyl-QS-17 2002 A component’, being triterpenoid             glycosides having a m/z of 2002 with negative mode             electrospray mass spectrometry. The desglucosyl-QS-17 2002 A             component corresponds to the A-isomer xylose chemotype and             may consist of desglucosyl-QS-17 2002 A:

-   -   -   ‘desglucosyl-QS-17 2148 A component’, being triterpenoid             glycosides having a m/z of 2148 with negative mode             electrospray mass spectrometry. The desglucosyl-QS-17 2148 A             component is believed to be identified in Kite 2004 as Peaks             70 and 72 and corresponds to the A-isomer rhamnose             chemotype. The desglucosyl-QS-17 2148 A component may             consist of desglucosyl-QS-17 2148 A V1 (i.e. apiose isomer):

-   -   and/or desglucosyl-QS-17 2148 A V2 (i.e. xylose isomer):

-   -   -   ‘desglucosyl-QS-17 2134 B component’, being triterpenoid             glycosides having a m/z of 2134 with negative mode             electrospray mass spectrometry. The desglucosyl-QS-17 2134 B             component is believed to be identified in Kite 2004 as Peak             67 and corresponds to B-isomers of the xylose chemotype. The             desglucosyl-QS-17 2134 B component may consist of             desglucosyl-QS-17 2134 B V1 (i.e. apiose isomer):

-   -   and/or desglucosyl-QS-17 2134 B V2 (i.e. xylose isomer):

-   -   -   ‘desglucosyl-QS-17 2002 B component’, being triterpenoid             glycosides having a m/z of 2002 with negative mode             electrospray mass spectrometry. The desglucosyl-QS-17 2002 B             component corresponds to the B-isomer of the xylose             chemotype and may consist of desglucosyl-QS-17 2002 B:

-   -   -   ‘desglucosyl-QS-17 2148 B component’, being triterpenoid             glycosides having a m/z of 2148 with negative mode             electrospray mass spectrometry. The desglucosyl-QS-17 2148 B             component is believed to be identified in Kite 2004 as Peak             65 and corresponds to B-isomers of the rhamnose chemotype.             The desglucosyl-QS-17 2148 B component may consist of             desglucosyl-QS-17 2148 B V1 (i.e. apiose isomer):

-   -   and/or desglucosyl-QS-17 2148 B V2 (i.e. xylose isomer):

-   -   QS-17 family components (i.e. triterpenoid glycosides having         beta-O-glucopyranosylation at the C3 position of the L-rhamnose         moiety and alpha-O-rhamnosylation at the C2 position of the         arabinofuranose moiety of QS-21 family components), such as:         -   ‘QS-17 2296 A component’, being the triterpenoid glycosides             identified as part of the QS-17 main peak in FIG. 2 and             having a m/z of 2296 with negative mode electrospray mass             spectrometry. The QS-17 2296 A component is believed to be             identified in Kite 2004 as Peak 59 and corresponds to the             A-isomers of xylose chemotype structure QS-Ill. The QS-17             2296 A component may consist of QS-17 2296 A V1 (i.e. apiose             isomer):

-   -   and/or QS-17 2296 A V2 (i.e. xylose isomer):

-   -   -   ‘QS-17 2164 A component’, being the triterpenoid glycosides             identified as part of the QS-17 main peak in FIG. 2 and             having a m/z of 2164 with negative mode electrospray mass             spectrometry. The QS-17 2164 A component is believed to be             identified in Kite 2004 as Peak 58 and corresponds to the             A-isomer xylose chemotype. The QS-17 2164 A component may             consist of QS-17 2164 A:

-   -   -   ‘QS-17 2310 A component’, being the triterpenoid glycosides             identified as part of the QS-17 main peak in FIG. 2 and             having a m/z of 2310 with negative mode electrospray mass             spectrometry. The QS-17 2310 A component is believed to be             identified in Kite 2004 as Peak 57 and corresponds to             A-isomers of the rhamnose chemotype. The QS-17 2310 A             component may consist of QS-17 2310 A V1 (i.e. apiose             isomer):

-   -   and/or QS-17 2310 A V2 (i.e. xylose isomer):

-   -   -   ‘QS-17 2296 B component’, being triterpenoid glycosides             having a m/z of 2296 with negative mode electrospray mass             spectrometry. The QS-17 2296 B component corresponds to the             B-isomers of xylose chemotype structure QS-III in Kite 2004.             The QS-17 2296 B component may consist of QS-17 2296 B V1             (i.e. apiose isomer):

-   -   and/or QS-17 2296 B V2 (i.e. xylose isomer):

-   -   -   ‘QS-17 2164 B component’, being triterpenoid glycosides             having a m/z of 2164 with negative mode electrospray mass             spectrometry. The QS-17 2164 B component corresponds to the             B-isomer xylose chemotype. The QS-17 2164 B component and             may consist of QS-17 2164 B:

-   -   -   ‘QS-17 2310 B component’, being triterpenoid having a m/z of             2310 with negative mode electrospray mass spectrometry. The             QS-17 2310 B component corresponds to B-isomers of the             rhamnose chemotype. The QS-17 2310 B component may consist             of QS-17 2310 B V1 (i.e. apiose isomer):

-   -   and/or QS-17 2310 B V2 (i.e. xylose isomer);

-   -   desarabinofuranosyl-QS-18 family components (i.e. triterpenoid         glycosides having beta-O-glucopyranosylation at the C3 position         of the L-rhamnose moiety and but lacking the arabinofuranose         moiety of QS-21 family components). The         desarabinofuranosyl-QS-18 family components are present in         relatively low amounts in extracts, meaning that they have not         been subjected to detailed characterisation.         Desarabinofuranosyl-QS-18 family components can be challenging         to isolate from QS-21 family components.         Desarabinofuranosyl-QS-18 family components include:         -   desarabinofuranosyl-QS-18 2018 A component (i.e.             triterpenoid glycosides identified as part of the ‘2018             Peak’ in FIG. 6 . Suitably the desarabinofuranosyl-QS-18             2018 A component in the UPLC-UV/MS methods described herein             has a retention time of approximately 4.5 min, the primary             component of the peak having a m/z of 2018 with negative             mode electrospray mass spectrometry. The             desarabinofuranosyl-QS-18 2018 A component may also be             identified in the UPLC-UV methods described herein with a             retention time of approximately 5.8 min. The             desarabinofuranosyl-QS-18 2018 A component is believed to be             identified in Kite 2004 as Peak 90 and corresponds to             A-isomers of the xylose chemotype. Putative structures have             been identified for the primary desarabinofuranosyl-QS-18             2018 A components using MS/MS. The desarabinofuranosyl-QS-18             2018 A component may consist of desarabinofuranosyl-QS-18             2018 A V1 (i.e. apiose isomer):

-   -   and/or desarabinofuranosyl-QS-18 2018 A V2 (i.e. xylose isomer):

-   -   -   desarabinofuranosyl-QS-18 1886 A component (i.e.             triterpenoid glycosides identified as part of the ‘2018             Peak’ in FIG. 6 . Suitably the desarabinofuranosyl-QS-18             1886 A component in the UPLC-UV/MS methods described herein             has a retention time of approximately 4.5 min and a m/z of             1886 with negative mode electrospray mass spectrometry. The             desarabinofuranosyl-QS-18 1886 A component and corresponds             to the A-isomer xylose chemotype may also be identified in             the UPLC-UV methods described herein with a retention time             of approximately 5.8 min. The desarabinofuranosyl-QS-18 1886             A component may consist of:

-   -   -   desarabinofuranosyl-QS-18 2032 A component (i.e.             triterpenoid glycosides identified as part of the ‘2018             Peak’ in FIG. 6 . Suitably the desarabinofuranosyl-QS-18             2032 A component in the UPLC-UV/MS methods described herein             has a retention time of approximately 4.5 min and a m/z of             2032 with negative mode electrospray mass spectrometry. The             desarabinofuranosyl-QS-18 2032 A component corresponds to             A-isomers of the rhamnose chemotype and may also be             identified in the UPLC-UV methods described herein with a             retention time of approximately 5.8 min. The             desarabinofuranosyl-QS-18 2032 A component may consist of             desarabinofuranosyl-QS-18 2032 A V1 (i.e. apiose isomer):

-   -   and/or desarabinofuranosyl-QS-18 2032 A V2 (i.e. xylose isomer):

-   -   -   desarabinofuranosyl-QS-18 2018 B component, i.e.             triterpenoid glycosides having a m/z of 2018 with negative             mode electrospray mass spectrometry. The             desarabinofuranosyl-QS-18 2018 B component corresponds to             B-isomers of the xylose chemotype. Desarabinofuranosyl-QS-18             2018 B component may consist of desarabinofuranosyl-QS-18             2018 B V1 (i.e. apiose isomer):

-   -   and/or desarabinofuranosyl-QS-18 2018 B V2 (i.e. xylose isomer):

-   -   desarabinofuranosyl-QS-18 1886 B component, i.e. triterpenoid         glycosides identified having a m/z of 1886 with negative mode         electrospray mass spectrometry. The desarabinofuranosyl-QS-18         1886 B component corresponds to the B-isomer xylose chemotype.         The desarabinofuranosyl-QS-18 1886 B component may consist of:

-   -   desarabinofuranosyl-QS-18 2032 B component, i.e. triterpenoid         glycosides having a m/z of 2032 with negative mode electrospray         mass spectrometry. The desarabinofuranosyl-QS-18 2032 B         component corresponds to B-isomers of the rhamnose chemotype.         The desarabinofuranosyl-QS-18 2032 B component may consist of         desarabinofuranosyl-QS-18 2032 B V1 (i.e. apiose isomer):

-   -   and/or desarabinofuranosyl-QS-18 2032 B V2 (i.e. xylose isomer):

-   -   acetylated desglucosyl-QS-17 family components (i.e.         triterpenoid glycosides having alpha-O-rhamnosylation at the C2         position of the arabinofuranose moiety and acetylation of the C3         position of the fucose of QS-21 family components), such as:         -   ‘acetylated desglucosyl-QS-17 2176 A component’, being             triterpenoid glycosides having a m/z of 2176 with negative             mode electrospray mass spectrometry. The acetylated             desglucosyl-QS-17 2176 A component corresponds to A-isomers             of the xylose chemotype. The acetylated desglucosyl-QS-17             2176 A component may consist of acetylated desglucosyl-QS-17             2176 A V1 (i.e. apiose isomer):

-   -   and/or acetylated desglucosyl-QS-17 2176 A V2 (i.e. xylose         isomer):

-   -   -   ‘acetylated desglucosyl-QS-17 2044 A component’, being             triterpenoid glycosides having m/z of 2044 with negative             mode electrospray mass spectrometry. The acetylated             desglucosyl-QS-17 2044 A component corresponds to the             A-isomer xylose chemotype. The acetylated desglucosyl-QS-17             2044 A component may consist of acetylated desglucosyl-QS-17             2044 A:

-   -   -   ‘acetylated desglucosyl-QS-17 2190 A component’, being             triterpenoid glycosides having a m/z of 2190 with negative             mode electrospray mass spectrometry. The acetylated             desglucosyl-QS-17 2190 A corresponds to the A-isomer             rhamnose chemotype. The acetylated desglucosyl-QS-17 2190 A             component may consist of acetylated desglucosyl-QS-17 2190 A             V1 (i.e. apiose isomer):

-   -   and/or acetylated desglucosyl-QS-17 2190 A V2 (i.e. xylose         isomer):

Starting saponins of direct relevance to the engineered glucosidase polypeptides are those having cleavable glucose residues, nevertheless, the engineered glucosidase polypeptides may be utilised in conjunction with additional enzymes capable of cleaving other sugar residues. Particular starting saponins of relevance to the engineered glucosidase polypeptides include:

-   -   QS-18 family components;     -   QS-17 family components; and     -   desarabinofuranosyl-QS-18 family components.

Starting saponins of direct relevance to the engineered rhamnosidase polypeptides are those having cleavable rhamnose residues, nevertheless, the engineered rhamnosidase polypeptides may be utilised in conjunction with additional enzymes capable to cleaving other sugar residues. Particular starting saponins of relevance to the engineered rhamnosidase polypeptides include:

-   -   desglucosyl-QS-17 family components;     -   QS-17 family components; and     -   acetylated desglucosyl-QS-17 family components.

The methods of the present invention enzymatically modify a starting saponin to provide a product saponin. The product saponin may be a naturally occurring saponin (i.e. a steroid or terpenoid glycoside found in nature, though the product saponin is itself obtained by the methods of the invention) or an artificially created saponin (i.e. a steroid or terpenoid glycoside not found in nature).

In some embodiments the product saponin is a steroid glycoside, in other embodiments the product saponin is a terpenoid glycoside, especially a triterpenoid glycoside.

Naturally occurring product saponins include those obtainable from plants of the genera Gypsophilia, Saponaria or Quillaja (Bomford, 1992). Especially of interest are product saponins obtainable from plants of Quillaja species. Particular product saponins of interest include those obtainable from Quillaja brasiliensis or Quillaja saponaria. In one embodiment the product saponin is obtainable from Quillaja saponaria. In one embodiment the product saponin is obtainable from Quillaja brasiliensis.

In certain embodiments the product saponin is a quillaic acid glycoside.

Product saponins obtainable from Quillaja saponaria include:

-   -   QS-18 family components (i.e. triterpenoid glycosides having         beta-O-glucopyranosylation at the C3 position of the L-rhamnose         moiety of QS-21 family components), such as:         -   ‘QS-18 2150 A component’. The QS-18 2150 A component may             consist of QS-18 2150 A V1 (i.e. apiose isomer):

-   -   and/or QS-18 2150 A V2 (i.e. xylose isomer):

-   -   -   ‘QS-18 2018 A component’. The QS-18 2018 A component may             consist of QS-18 2018 A:

-   -   -   ‘QS-18 2164 A component’. The QS-18 2164 A component may             consist of QS-18 2164 A V1 (i.e. apiose isomer):

-   -   and/or QS-18 2164 A V2 (i.e. xylose isomer):

-   -   -   ‘QS-18 2150 B component’. The QS-18 2150 B component may             consist of QS-18 2150 B V1 (i.e. apiose isomer):

-   -   and/or QS-18 2150 B V2 (i.e. xylose isomer):

-   -   -   ‘QS-18 2018 B component’. The QS-18 2018 B component may             consist of QS-18 2018 B:

-   -   -   ‘QS-18 2164 B component’. The QS-18 2164 B component may             consist of QS-18 2164 B V1 (i.e. apiose isomer):

-   -   and/or QS-18 2164 B V2 (i.e. xylose isomer):

-   -   desglucosyl-QS-17 family components (i.e. triterpenoid         glycosides having alpha-O-rhamnosylation at the C2 position of         the arabinofuranose moiety of QS-21 family components), such as:         -   ‘desglucosyl-QS-17 2134 A component’. The desglucosyl-QS-17             2134 A component may consist of desglucosyl-QS-17 2134 A V1             (i.e. apiose isomer):

-   -   and/or desglucosyl-QS-17 2134 A V2 (i.e. xylose isomer):

-   -   -   ‘desglucosyl-QS-17 2002 A component’. The desglucosyl-QS-17             2002 A component may consist of desglucosyl-QS-17 2002 A:

-   -   -   ‘desglucosyl-QS-17 2148 A component’. The desglucosyl-QS-17             2148 A component may consist of desglucosyl-QS-17 2148 A V1             (i.e. apiose isomer):

-   -   and/or desglucosyl-QS-17 2148 A V2 (i.e. xylose isomer):

-   -   ‘desglucosyl-QS-17 2134 B component’. The desglucosyl-QS-17 2134         B component may consist of desglucosyl-QS-17 2134 B V1 (i.e.         apiose isomer):

-   -   and/or desglucosyl-QS-17 2134 B V2 (i.e. xylose isomer):

-   -   -   ‘desglucosyl-QS-17 2002 B component’. The desglucosyl-QS-17             2002 B component may consist of desglucosyl-QS-17 2002 B:

-   -   -   ‘desglucosyl-QS-17 2148 B component’. The desglucosyl-QS-17             2148 B component may consist of desglucosyl-QS-17 2148 B V1             (i.e. apiose isomer):

-   -   and/or desglucosyl-QS-17 2148 B V2 (i.e. xylose isomer):

-   -   QS-21 family components, such as:         -   ‘QS-21 1988 A component’, being the triterpenoid glycosides             identified as part of the QS-21 main peak in FIG. 6 and             having a m/z of 1988 with negative mode electrospray mass             spectrometry. Suitably the QS-21 1988 A component in the             UPLC-UV/MS methods described herein has a retention time of             approximately 4.4 min and a m/z of 1988 with negative mode             electrospray mass spectrometry. The QS-21 1988 A component             is believed to be identified in Kite 2004 as Peak 88 and             corresponds to the A-isomer xylose chemotype structures S6             (apiose isomer) and S4 (xylose isomer) characterised in             Nyberg 2000 and Nyberg 2003. The QS-21 1988 A component may             consist of QS-21 1988 A V1 (i.e. apiose isomer):

-   -   and QS-21 1988 A V2 (i.e. xylose isomer):

-   -   -   ‘QS-21 1856 A component’, being the triterpenoid glycosides             identified as part of the QS-21 main peak in FIG. 6 and             having a m/z of 1856 with negative mode electrospray mass             spectrometry. Suitably the QS-21 1856 A component in the             UPLC-UV/MS methods described herein has a retention time of             approximately 4.4 min and a m/z of 1856 with negative mode             electrospray mass spectrometry. The QS-21 1856 A component             is believed to be identified in Kite 2004 as Peak 86 and             corresponds to the A-isomer xylose chemotype structure S2             characterised in Nyberg 2000 and Nyberg 2003. The QS-21 1856             A component may consist of:

-   -   -   ‘QS-21 2002 A component’, being the triterpenoid glycosides             identified as part of the QS-21 main peak in FIG. 6 and             having a m/z of 2002 with negative mode electrospray mass             spectrometry. Suitably the QS-21 2002 A component in the             UPLC-UV/MS methods described herein has a retention time of             approximately 4.4 min and a m/z of 2002 with negative mode             electrospray mass spectrometry. The QS-21 2002 A component             is believed to be identified in Kite 2004 as Peak 85 and             corresponds to the A-isomer rhamnose chemotype of structures             S3 and S5 characterised in Nyberg 2000 and Nyberg 2003. The             QS-21 2002 A component may consist of QS-21 2002 A V1 (i.e.             apiose isomer):

-   -   and QS-21 2002 A V2 (i.e. xylose isomer):

-   -   -   ‘QS-21 1988 B component’, being the triterpenoid glycosides             identified as part of the B-isomer peak in FIG. 6 and having             a m/z of 1988 with negative mode electrospray mass             spectrometry. Suitably the QS-21 1988 B component in the             UPLC-UV/MS methods described herein has a retention time of             approximately 4.0 min and a m/z of 1988 with negative mode             electrospray mass spectrometry. The QS-21 1988 B component             corresponds to the B-isomer xylose chemotype structures S6a             (apiose isomer) and S4a (xylose isomer) characterised in             Nyberg 2000 and Nyberg 2003. The QS-21 1988 B component may             consist of QS-21 1988 B V1 (i.e. apiose isomer):

-   -   and QS-21 1988 B V2 (i.e. xylose isomer):

-   -   -   ‘QS-21 1856 B component’, being the triterpenoid glycosides             identified as part of the B-isomer peak in FIG. 6 and having             a m/z of 1856 with negative mode electrospray mass             spectrometry. The QS-21 1856 B component corresponds to the             B-isomer xylose chemotype structure S2a characterised in             Nyberg 2000 and Nyberg 2003. The QS-21 1856 B component may             consist of:

-   -   -   ‘QS-21 2002 B component’, being the triterpenoid glycosides             having a m/z of 2002 with negative mode electrospray mass             spectrometry. The QS-21 2002 B component corresponds to the             B-isomer rhamnose chemotype of structures S3a and S5a             characterised in Nyberg 2000 and Nyberg 2003. The QS-21 2002             B component may consist of QS-21 2002 B component V1 (i.e.             apiose isomer):

-   -   and QS-21 2002 B V2 (i.e. xylose isomer):

-   -   desarabinofuranosyl-QS-21 family components (i.e. triterpenoid         glycosides lacking the arabinofuranose moiety of QS-21 family         components). The desarabinofuranosyl-QS-21 family components are         present in relatively low amounts in extracts, meaning that they         have not been subjected to detailed characterisation.         Desarabinofuranosyl-QS-21 family components include:         -   desarabinofuranosyl-QS-21 1856 A component (i.e.             triterpenoid glycosides identified as part of the             ‘Lyophilization Peak’ in FIG. 6 . Suitably the             desarabinofuranosyl-QS-21 1856 A component in the UPLC-UV/MS             methods described herein has a retention time of             approximately 4.7 min, the primary component of the peak             having a m/z of 1856 with negative mode electrospray mass             spectrometry. The desarabinofuranosyl-QS-21 1856 A component             is believed to be identified in Kite 2004 as Peak 96.             Putative structures have been identified for the primary             desarabinofuranosyl-QS-21 1856 A components using MS/MS. The             desarabinofuranosyl-QS-21 1856 A component may consist of             desarabinofuranosyl-QS-21 1856 A V1 (i.e. apiose isomer):

-   -   and/or desarabinofuranosyl-QS-21 1856 A V2 (i.e. xylose isomer):

-   -   -   desarabinofuranosyl-QS-21 1712 A component (i.e.             triterpenoid glycosides identified as part of the             ‘Lyophilization Peak’ in FIG. 6 . Suitably the             desarabinofuranosyl-QS-21 1712 A component in the UPLC-UV/MS             methods described herein has a retention time of             approximately 4.7 min and a m/z of 1712 with negative mode             electrospray mass spectrometry. The             desarabinofuranosyl-QS-21 1712 A component may consist of             desarabinofuranosyl-QS-21 1712 A:

-   -   -   desarabinofuranosyl-QS-21 1870 A component, i.e.             triterpenoid glycosides having a m/z of 1870 with negative             mode electrospray mass spectrometry. The             desarabinofuranosyl-QS-21 1870 A component may consist of             desarabinofuranosyl-QS-21 1870 A V1 (i.e. apiose isomer):

-   -   and/or desarabinofuranosyl-QS-21 1870 A V2 (i.e. xylose isomer):

-   -   -   desarabinofuranosyl-QS-21 1856 B component, i.e.             triterpenoid glycosides having a m/z of 1856 with negative             mode electrospray mass spectrometry. The             desarabinofuranosyl-QS-21 1856 B component may consist of             desarabinofuranosyl-QS-21 1856 B V1 (i.e. apiose isomer):

-   -   and/or desarabinofuranosyl-QS-21 1856 B V2 (i.e. xylose isomer):

-   -   -   desarabinofuranosyl-QS-21 1712 B component, i.e.             triterpenoid glycosides having a m/z of 1712 with negative             mode electrospray mass spectrometry. The             desarabinofuranosyl-QS-21 1712 B component may consist of             desarabinofuranosyl-QS-21 1712 B:

-   -   -   desarabinofuranosyl-QS-21 1870 B component, i.e.             triterpenoid glycosides having a m/z of 1870 with negative             mode electrospray mass spectrometry. The             desarabinofuranosyl-QS-21 1870 B component may consist of             desarabinofuranosyl-QS-21 1870 B V1 (i.e. apiose isomer):

-   -   and/or desarabinofuranosyl-QS-21 1870 B V2 (i.e. xylose isomer):

-   -   acetylated QS-21 family components (i.e. triterpenoid glycosides         having acetylation of the C3 position of the fucose of QS-21         family components), such as:         -   ‘acetylated QS-21 2030 A component’, being triterpenoid             glycosides having a m/z of 2030 with negative mode             electrospray mass spectrometry. The acetylated QS-21 2030 A             corresponds to the A-isomer xylose chemotype. The acetylated             QS-21 2030 A component may consist of acetylated QS-21 2030             A V1 (i.e. apiose isomer):

-   -   and/or acetylated QS-21 2030 A V2 (i.e. xylose isomer):

-   -   -   ‘acetylated QS-21 1898 A component’, being triterpenoid             glycosides having a m/z of 1898 with negative mode             electrospray mass spectrometry. The acetylated QS-21 1898 A             corresponds to the A-isomer xylose chemotype. The acetylated             QS-21 1898 A component may consist of acetylated QS-21 1898             A:

-   -   -   ‘acetylated QS-21 2044 A component’, being triterpenoid             glycosides having a m/z of 2044 with negative mode             electrospray mass spectrometry. The acetylated QS-21 2044 A             corresponds to A-isomers of the rhamnose chemotype. The             acetylated QS-21 2044 A component may consist of acetylated             QS-21 2044 A V1 (i.e. apiose isomer):

-   -   and/or acetylated QS-21 2044 A V2 (i.e. xylose isomer):

Product saponins of direct relevance to the engineered glucosidase polypeptides are those where a glucose residue has been cleaved relative to a starting saponin. Nevertheless, the engineered glucosidase polypeptides may be utilised in conjunction with additional enzymes capable to cleaving other sugar residues. Particular product saponins of relevance to the engineered glucosidase polypeptides include:

-   -   desglucosyl-QS-17 family components;     -   QS-21 family components; and     -   desarabinofuranosyl-QS-21 family components.

Product saponins of direct relevance to the engineered rhamnosidase polypeptides are those where a rhamnose residue has been cleaved relative to a starting saponin. Nevertheless, the engineered rhamnosidase polypeptides may be utilised in conjunction with additional enzymes capable to cleaving other sugar residues. Particular product saponins of relevance to the engineered rhamnosidase polypeptides include:

-   -   QS-18 family components;     -   QS-21 family components; and     -   acetylated QS-21 family components.

The term QS-18 family components as used herein means the xylose chemotype QS-18 2150 component (A and B isomers, and apiose and xylose isomers: QS-18 2150 A V1, QS-18 2150 A V2, QS-18 2150 B V1 and QS-18 2150 B V2), the xylose chemotype QS-18 2018 component (A and B isomers: QS-18 2018 A and QS-18 2018 B), the rhamnose chemotype QS-18 2164 component (A and B isomers, and apiose and xylose isomers: QS-18 2164 A V1, QS-18 2164 A V2, QS-18 2164 B V1 and QS-18 2164 B V2).

The term desglucosyl-QS-17 family components as used herein means the xylose chemotype desglucosyl-QS-17 2134 component (A and B isomers, and apiose and xylose isomers: desglucosyl-QS-17 2134 A V1, desglucosyl-QS-17 2134 A V2, desglucosyl-QS-17 2134 B V1 and desglucosyl-QS-17 2134 B V2), the xylose chemotype desglucosyl-QS-17 2002 component (A and B isomers: desglucosyl-QS-17 2002 A and desglucosyl-QS-17 2002 B), the rhamnose chemotype desglucosyl-QS-17 2148 component (A and B isomers, and apiose and xylose isomers: desglucosyl-QS-17 2148 A V1, desglucosyl-QS-17 2148 A V2, desglucosyl-QS-17 2148 B V1 and desglucosyl-QS-17 2148 B V2).

The term QS-17 family components as used herein means the xylose chemotype QS-17 2296 component (A and B isomers, and apiose and xylose isomers: QS-17 2296 A V1, QS-17 2296 A V2, QS-17 2296 B V1 and QS-17 2296 B V2), the xylose chemotype QS-17 2164 component (A and B isomers: QS-17 2164 A and QS-17 2164 B), the rhamnose chemotype QS-17 2310 component (A and B isomers, and apiose and xylose isomers: QS-17 2310 A V1, QS-17 2310 A V2, QS-17 2310 B V1 and QS-17 2310 B V2).

The term QS-21 family components as used herein means the xylose chemotype QS-21 1988 component (A and B isomers, and apiose and xylose isomers: QS-21 1988 A V1, QS-21 1988 A V2, QS-21 1988 B V1 and QS-21 1988 B V2), the xylose chemotype QS-21 1856 component (A and B isomers: QS-21 1856 A and QS-21 1856 B), the rhamnose chemotype QS-21 2002 component (A and B isomers, and apiose and xylose isomers: QS-21 2002 A V1, QS-21 2002 A V2, QS-21 2002 B V1 and QS-21 2002 B V2).

The term desarabinofuranosyl-QS-18 family components as used herein means the xylose chemotype desarabinofuranosyl-QS-18 2018 component (A and B isomers, and apiose and xylose isomers: desarabinofuranosyl-QS-18 2018 A V1, desarabinofuranosyl-QS-18 2018 A V2, desarabinofuranosyl-QS-18 2018 B V1 and desarabinofuranosyl-QS-18 2018 B V2), the xylose chemotype desarabinofuranosyl-QS-18 1886 component (A and B isomers: desarabinofuranosyl-QS-18 1886 A and desarabinofuranosyl-QS-18 1886 B), the rhamnose chemotype desarabinofuranosyl-QS-18 2032 component (A and B isomers, and apiose and xylose isomers: desarabinofuranosyl-QS-18 2032 A V1, desarabinofuranosyl-QS-18 2032 A V2, desarabinofuranosyl-QS-18 2032 B V1 and desarabinofuranosyl-QS-18 2032 B V2).

The term acetylated desglucosyl-QS-17 family components as used herein means xylose chemotype acetylated desglucosyl-QS-17 2176 component (apiose and xylose isomers: acetylated desglucosyl-QS-17 2176 A V1 and acetylated desglucosyl-QS-17 2176 A V2), the xylose chemotype acetylated desglucosyl-QS-17 2044 A component, the rhamnose chemotype acetylated desglucosyl-QS-17 2190 component (apiose and xylose isomers: acetylated desglucosyl-QS-17 2190 A V1 and acetylated desglucosyl-QS-17 2190 A V2).

The term desarabinofuranosyl-QS-21 family components as used herein means xylose chemotype desarabinofuranosyl-QS-21 1856 component (A and B isomers, and apiose and xylose isomers: desarabinofuranosyl-QS-21 1856 A V1, desarabinofuranosyl-QS-21 1856 A V2, desarabinofuranosyl-QS-21 1856 B V1 and desarabinofuranosyl-QS-21 1856 B V2), the xylose chemotype desarabinofuranosyl-QS-21 1712 component (A and B isomers: desarabinofuranosyl-QS-21 1712 A and desarabinofuranosyl-QS-21 1712 B), the rhamnose chemotype desarabinofuranosyl-QS-21 1870 component (A and B isomers, and apiose and xylose isomers: desarabinofuranosyl-QS-21 1870 A V1, desarabinofuranosyl-QS-21 1870 A V2, desarabinofuranosyl-QS-21 1870 B V1 and desarabinofuranosyl-QS-21 1870 B V2).

The term acetylated QS-21 family components as used herein means xylose chemotype acetylated QS-21 2030 component (apiose and xylose isomers: acetylated QS-21 2030 A V1 and acetylated QS-21 2030 A V2), the xylose chemotype acetylated QS-21 1898 A component, the rhamnose chemotype acetylated QS-21 2044 component (apiose and xylose isomers: acetylated QS-21 2044 A V1 and acetylated QS-21 2044 A V2).

Starting Materials

Suitably a starting saponin is obtained by extraction from a starting material. The starting material may be plant material obtained from plants of the genera Gypsophilia, Saponaria or Quillaja (Bomford, 1992), such as plant material obtained from plants of Quillaja species. Particular plant material includes that obtained from Quillaja brasiliensis or Quillaja saponaria. In one embodiment the plant material is obtained from Quillaja saponaria. In one embodiment the plant material is obtained from Quillaja brasiliensis.

Extraction may be from complete plants. Alternatively, extraction may be from selected plant tissues. Extraction from selected plant tissues may be from plant material including wood or bark, such as from plant material which is wood or bark. In some embodiments, extraction is from plant material including bark, such as from plant material which is bark.

Extraction may be from plant material obtained from an adult plant. Alternatively, extraction may be from plant material obtained from a young plant, such as plants of less than 5 years old, such as less than 3 years old. (Schlotterbeck, 2015; WO2018057031)

Extraction may be performed using water or lower alcohols (e.g. methanol or ethanol) as solvents, including mixtures thereof. In one embodiment the starting saponin is obtained by aqueous extraction (e.g. using solvent comprising at least 80% v/v water, especially at least 90% v/v water, such as at least 95% v/v water). In one embodiment the starting saponin is obtained by methanol extraction (e.g. using solvent comprising at least 80% v/v methanol, especially at least 90% v/v methanol, such as at least 95% v/v methanol). In one embodiment the starting saponin is obtained by ethanol extraction (e.g. using solvent comprising at least 80% v/v ethanol, especially at least 90% v/v ethanol, such as at least 95% v/v ethanol). In one embodiment the starting saponin is obtained by methanol/ethanol extraction (e.g. using solvent comprising at least 20% v/v methanol, especially at least 30% v/v methanol, such as at least 40% v/v methanol and at least 20% ethanol, especially at least 30% v/v ethanol, such as at least 40% v/v ethanol). In one embodiment the starting saponin is obtained by water/ethanol extraction (e.g. using solvent comprising at least 20% v/v water, especially at least 30% v/v water, such as at least 40% v/v water and at least 20% ethanol, especially at least 30% v/v ethanol, such as at least 40% v/v ethanol. In one embodiment the starting saponin is obtained by water/methanol extraction (e.g. using solvent comprising at least 20% v/v water, especially at least 30% v/v water, such as at least 40% v/v water and at least 20% methanol, especially at least 30% v/v methanol, such as at least 40% v/v methanol).

Methods of the invention may be applied to starting saponin in a range of contexts. A starting saponin may be in the form of a minor component in a saponin containing composition (ignoring solvents, if any), such as a minor component of a plant material extract. A starting saponin may be in the form of a major component in a saponin containing composition, such as a major component in a plant material extract. A starting saponin may be in the form of a minor component in a processed, such as partially purified, plant material extract. A starting saponin may be in the form of a major component in a processed, such as partially purified, plant material extract. In some embodiments the starting saponin is substantially purified at the time of enzymatic modification.

Purification refers to the isolation of a component from other components. Partial purification therefore means the isolation of a components, to some degree, from other components. Substantial purification means the substantial isolation of a component from other components, such as wherein the component comprises at least 50% w/w, especially as at least 70%, particularly at least 80%, for example at least 90% of the component content (50%, 70%, 80% and 90% purity, respectively). Partial purification, in relation to an extract, means the isolation of the starting saponin, to some degree, from other extracted components. Substantially purified, in relation to an extract, means the substantial isolation of the starting saponin from other extracted components, such as wherein the starting saponin comprises at least 50% w/w, especially as at least 70%, particularly at least 80%, for example at least 90% of the extracted component content. Partial or substantial purification can be undertaken through various means including chromatography, filtration over semi-permeable membranes, treatment with selective adsorbants such as polyvinylpolypyrrolidone (PVPP) and the like.

Although a starting saponin may be a specific chemical entity, in many circumstances involving saponins obtained by extraction a plurality of starting saponins may be present, these being enzymatically modified to provide their corresponding product saponins. As mentioned above for individual saponins, the invention may be applied to a plurality of starting saponins in a range of contexts mutatis mutandis. A plurality of starting saponins comprising related starting saponins may undergo equivalent enzymatic modification concurrently. A plurality of starting saponins comprising distinguishable starting saponins may undergo different enzymatic modifications concurrently (in the presence of more than one enzyme) or in series (sequential treatment with separate enzymes). A plurality of starting saponins may contain both related and distinguishable starting saponins.

Methods of the invention may be applied to a starting saponin in the form of a component of:

-   -   crude extract, such as water and/or lower alcohol extract,         especially aqueous extract;     -   crude bark extract (CBE), such as water and/or lower alcohol         bark extract, especially aqueous bark extract;     -   partially purified extract, such as water and/or lower alcohol         extract, especially aqueous extract;     -   partially purified bark extract, such as water and/or lower         alcohol bark extract, especially aqueous bark extract;     -   PVPP treated extract, such as PVPP treated water and/or lower         alcohol extract, especially PVPP treated aqueous extract;     -   PVPP treated bark extract (TBE), such as PVPP treated water         and/or lower alcohol bark extract, especially PVPP treated         aqueous bark extract;     -   Quil A;     -   Fraction A;     -   Fraction B (see Nyberg 2003);     -   Fraction C;     -   QS-7;     -   QS-17;     -   QS-18; or     -   QS-21.

Methods of the invention may be applied to a starting saponin in a composition comprising:

-   -   QS-7 family components and QS-18 family components;     -   QS-7 family components and QS-17 family components;     -   QS-17 family components and QS-18 family components;     -   QS-7 family components, QS-17 family components and QS-18 family         components.

As with other QS families, the QS-7 family components contains a plurality of related structures including xylose and rhamnose chemotypes, xylose and apiose isomers, A and B isomers:

Certain QS-7 family compounds may lack glucose, or the rhamnose attached to the beta-D-fuc.

Enzymatic Modifications

The present invention provides the enzymatic modification of saponins. Enzymatic modifications envisaged in the present invention include the conversion of a starting saponin into a product saponin by the removal of one or more sugar residues from the starting saponin. Suitably the enzymatic modifications envisaged in the present invention are the conversion of a starting saponin into a product saponin by the removal of one or more sugar residues from the starting saponin.

In certain embodiments the enzymatic modification involves the removal of a single sugar residue i.e. removal of a terminal sugar residue (‘exo’ action) from a starting saponin. In other embodiments enzymatic conversion involves the removal of a plurality of sugar residues from a starting saponin i.e. cleavage at a saccharide linkage other than in a terminal location (‘endo’ action), resulting in removal of a plurality of sugar residues (such as 2, 3 or 4 sugar residues) attached through said saccharide linkage.

Particular sugar residues which may be removed comprise (such as consist of):

-   -   glucose, in particular a terminal glucose, especially a         beta-glucose, such as a beta-glucose from a quillaic acid         glycoside, for example the beta-D-glucose residue highlighted         below:     -   rhamnose, in particular a terminal rhamnose, especially an         alpha-rhamnose, such as an alpha-rhamnose from a quillaic acid         glycoside, for example the alpha-L-rhamnose residues highlighted         below:     -   in particular

Particular single sugar enzymatic conversions of interest include:

-   -   QS-18 family components to QS-21 family components, such as:         -   QS-18 2150 component (i.e. QS-18 2150 A and/or QS-18 2150 B)             to QS-21 1988 component, such as:             -   QS-18 2150 A component to QS-21 1988 A component, such                 as:                 -   QS-18 2150 A V1 to QS-21 1988 A V1                 -   QS-18 2150 A V2 to QS-21 1988 A V2             -   QS-18 2150 B component to QS-21 1988 B component, such                 as:                 -   QS-18 2150 B V1 to QS-21 1988 B V1                 -   QS-18 2150 B V2 to QS-21 1998 B V2             -   QS-18 2150 V1 component (i.e. QS-18 2150 A V1 and/or                 QS-18 2150 B V1) to QS-21 1988 V1 component, such as:                 -   QS-18 2150 A V1 to QS-21 1988 A V1                 -   QS-18 2150 B V1 to QS-21 1988 B V1             -   QS-18 2150 V2 component (i.e. QS-18 2150 A V2 and/or                 QS-18 2150 B V2) to QS-21 1988 V2 component, such as:                 -   QS-18 2150 A V2 to QS-21 1988 A V2                 -   QS-18 2150 B V2 to QS-21 1988 B V2         -   QS-18 2018 component (i.e. QS-18 2018 A and/or QS-18 2018 B)             to QS-21 1856 component, such as:             -   QS-18 2018 A component to QS-21 1856 A component             -   QS-18 2018 B component to QS-21 1856 B component         -   QS-18 2164 component (i.e. QS-18 2164 A and/or QS-18 2164 B)             to QS-21 2002 component, such as:             -   QS-18 2164 A component to QS-21 2002 A component, such                 as:                 -   QS-18 2164 A V1 to QS-21 2002 A V1                 -   QS-18 2164 A V2 to QS-21 2002 A V2             -   QS-18 2164 B component to QS-21 2002 B component, such                 as:                 -   QS-18 2164 B V1 to QS-21 2002 B V1                 -   QS-18 2164 B V2 to QS-21 2002 B V2             -   QS-18 2164 V1 component (i.e. QS-18 2164 A V1 and/or                 QS-18 2164 B V1) to QS-21 2002 V1 component, such as:                 -   QS-18 2164 A V1 to QS-21 2002 A V1                 -   QS-18 2164 B V1 to QS-21 2002 B V1             -   QS-18 2164 V2 component (i.e. QS-18 2164 A V2 and/or                 QS-18 2164 B V2) to QS-21 2002 V2 component, such as:                 -   QS-18 2164 A V2 to QS-21 2002 A V2                 -   QS-18 2164 B V2 to QS-21 2002 B V2     -   desglucosyl-QS-17 family components to QS-21 family components,         such as:         -   desglucosyl-QS-17 2134 component (i.e. desglucosyl-QS-17             2134 A and/or desglucosyl-QS-17 2134 B) to QS-21 1988             component, such as:             -   desglucosyl-QS-17 2134 A component to QS-21 1988 A                 component, such as:                 -   desglucosyl-QS-17 2134 A V1 to QS-21 1988 A V1                 -   desglucosyl-QS-17 2134 A V2 to QS-21 1988 A V2             -   desglucosyl-QS-17 2134 B component to QS-21 1988 B                 component, such as:                 -   desglucosyl-QS-17 2134 B V1 to QS-21 1988 B V1                 -   desglucosyl-QS-17 2134 B V2 to QS-21 1988 B V2             -   desglucosyl-QS-17 2134 V1 component (i.e.                 desglucosyl-QS-17 2134 A V1 and/or desglucosyl-QS-17                 2134 B V1) to QS-21 1988 V1 component, such as:                 -   desglucosyl-QS-17 2134 A V1 to QS-21 1988 A V1                 -   desglucosyl-QS-17 2134 B V1 to QS-21 1988 B V1             -   desglucosyl-QS-17 2134 V2 component (i.e.                 desglucosyl-QS-17 2134 A V2 and/or desglucosyl-QS-17                 2134 B V2) to QS-21 1988 V2 component, such as:                 -   desglucosyl-QS-17 2134 A V2 to QS-21 1988 A V2                 -   desglucosyl-QS-17 2134 B V2 to QS-21 1988 B V2         -   desglucosyl-QS-17 2002 component (i.e. desglucosyl-QS-17             2002 A and/or desglucosyl-QS-17 2002 B) to QS-21 1856             component, such as:             -   desglucosyl-QS-17 2002 A component to QS-21 1856 A                 component             -   desglucosyl-QS-17 2002 B component to QS-21 1856 B                 component         -   desglucosyl-QS-17 2148 component (i.e. desglucosyl-QS-17             2148 A and/or desglucosyl-QS-17 2148 B) to QS-21 2002             component, such as:             -   desglucosyl-QS-17 2148 A component to QS-21 2002 A                 component, such as:                 -   desglucosyl-QS-17 2148 A V1 to QS-21 2002 A V1                 -   desglucosyl-QS-17 2148 A V2 to QS-21 2002 A V2             -   desglucosyl-QS-17 2148 B component to QS-21 2002 B                 component, such as:                 -   desglucosyl-QS-17 2148 B V1 to QS-21 2002 B V1                 -   desglucosyl-QS-17 2148 B V2 to QS-21 2002 B V2             -   desglucosyl-QS-17 2134 V1 component (i.e.                 desglucosyl-QS-17 2134 A V1 and/or desglucosyl-QS-17                 2134 B V1) to QS-21 1988 V1 component, such as:                 -   desglucosyl-QS-17 2148 A V1 to QS-21 2002 A V1                 -   desglucosyl-QS-17 2148 B V1 to QS-21 2002 B V1             -   desglucosyl-QS-17 2134 V2 component (i.e.                 desglucosyl-QS-17 2134 A V2 and/or desglucosyl-QS-17                 2134 B V2) to QS-21 1988 V1 component, such as:                 -   desglucosyl-QS-17 2148 A V2 to QS-21 2002 A V2                 -   desglucosyl-QS-17 2148 B V2 to QS-21 2002 B V2     -   QS-17 family components to QS-18 family components, such as:         -   QS-17 2296 component (i.e. QS-17 2296 A and/or QS-17 2296 B)             to QS-18 2150 component, such as:             -   QS-17 2296 A component to QS-18 2150 A component, such                 as:                 -   QS-17 2296 A V1 to QS-18 2150 A V1                 -   QS-17 2296 A V2 to QS-18 2150 A V2             -   QS-17 2296 B component to QS-18 2150 B component, such                 as:                 -   QS-17 2296 B V1 to QS-18 2150 B V1                 -   QS-17 2296 B V2 to QS-18 2150 B V2             -   QS-17 2296 V1 component (i.e. QS-17 2296 A V1 and/or                 QS-17 2296 B V1) to QS-18 2150 V1 component, such as:                 -   QS-17 2296 A V1 to QS-18 2150 A V1                 -   QS-17 2296 B V1 to QS-18 2150 B V1             -   QS-17 2296 V2 component (i.e. QS-17 2296 A V2 and/or                 QS-17 2296 B V1) to QS-18 2150 V2 component, such as:                 -   QS-17 2296 A V2 to QS-18 2150 A V2                 -   QS-17 2296 B V2 to QS-18 2150 B V2         -   QS-17 2164 component (i.e. QS-17 2164 A and/or QS-17 2164 B)             to QS-18 2018 component, such as:             -   QS-17 2164 A component to QS-18 2018 A component             -   QS-17 2164 B component to QS-18 2018 B component         -   QS-17 2310 component (i.e. QS-17 2310 A and/or QS-17 2310 B)             to QS-18 2164 component, such as:             -   QS-17 2310 A component to QS-18 2164 A component, such                 as:                 -   QS-17 2310 A V1 to QS-18 2164 A V1                 -   QS-17 2310 A V2 to QS-18 2164 A V2             -   QS-17 2310 B component to QS-18 2164 B component, such                 as:                 -   QS-17 2310 B V1 to QS-18 2164 B V1                 -   QS-17 2310 B V2 to QS-18 2164 B V2             -   QS-17 2310 V1 component (i.e. QS-17 2310 A V1 and/or                 QS-17 2310 B V1) to QS-18 2164 V1 component, such as:                 -   QS-17 2310 A V1 to QS-18 2164 A V1                 -   QS-17 2310 B V1 to QS-18 2164 B V1             -   QS-17 2310 V2 component (i.e. QS-17 2310 A V2 and/or                 QS-17 2310 B V2) to QS-18 2164 V2 component, such as:                 -   QS-17 2310 A V2 to QS-18 2164 A V2                 -   QS-17 2310 B V2 to QS-18 2164 B V2     -   QS-17 family components to desglucosyl-QS-17 family components,         such as:         -   QS-17 2296 component (i.e. QS-17 2296 A and/or QS-17 2296 B)             to desglucosyl-QS-17 2134 component, such as:             -   QS-17 2296 A component to desglucosyl-QS-17 2134 A                 component, such as:                 -   QS-17 2296 A V1 to desglucosyl-QS-17 2134 A V1                 -   QS-17 2296 A V2 to desglucosyl-QS-17 2134 A V2             -   QS-17 2296 B component to desglucosyl-QS-17 2134 B                 component, such as:                 -   QS-17 2296 B V1 to desglucosyl-QS-17 2134 B V1                 -   QS-17 2296 B V2 to desglucosyl-QS-17 2134 B V2             -   QS-17 2296 V1 component (i.e. QS-17 2296 A V1 and/or                 QS-17 2296 B V1) to desglucosyl-QS-17 2134 V1 component,                 such as:                 -   QS-17 2296 A V1 to desglucosyl-QS-17 2134 A V1                 -   QS-17 2296 B V1 to desglucosyl-QS-17 2134 B V1             -   QS-17 2296 V2 component (i.e. QS-17 2296 A V2 and/or                 QS-17 2296 B V1) to desglucosyl-QS-17 2134 V2 component,                 such as:                 -   QS-17 2296 A V2 to desglucosyl-QS-17 2134 A V2                 -   QS-17 2296 B V2 to desglucosyl-QS-17 2134 B V2         -   QS-17 2164 component (i.e. QS-17 2164 A and/or QS-17 2164 B)             to desglucosyl-QS-17 2002 component, such as:             -   QS-17 2164 A component to desglucosyl-QS-17 2002 A             -   QS-17 2164 B component to desglucosyl-QS-17 2002 B         -   QS-17 2310 component (i.e. QS-17 2310 A and/or QS-17 2310 B)             to desglucosyl-QS-17 2148 component, such as:             -   QS-17 2310 A component to desglucosyl-QS-17 2148 A                 component, such as:                 -   QS-17 2310 A V1 to desglucosyl-QS-17 2148 A V1                 -   QS-17 2310 A V2 to desglucosyl-QS-17 2148 A V2             -   QS-17 2310 B component to QS-21, such as:                 -   QS-17 2310 B V1 to desglucosyl-QS-17 2148 B V1                 -   QS-17 2310 B V2 to desglucosyl-QS-17 2148 B V2             -   QS-17 2310 V1 component (i.e. QS-17 2310 A V1 and/or                 QS-17 2310 B V1) to desglucosyl-QS-17 2148 V1 component,                 such as:                 -   QS-17 2310 A V1 to desglucosyl-QS-17 2148 A V1                 -   QS-17 2310 B V1 to desglucosyl-QS-17 2148 B V1             -   QS-17 2310 V2 component (i.e. QS-17 2310 A V2 and/or                 QS-17 2310 B V2) to desglucosyl-QS-17 2148 V2 component,                 such as:                 -   QS-17 2310 A V2 to desglucosyl-QS-17 2148 A V2                 -   QS-17 2310 B V2 to desglucosyl-QS-17 2148 B V2                     Other single sugar enzymatic conversions of interest                     include:     -   desarabinofuranosyl-QS-18 family components to         desarabinofuranosyl-QS-21 family components, such as:         -   desarabinofuranosyl-QS-18 2018 component (i.e.             desarabinofuranosyl-QS-18 2018 A and/or             desarabinofuranosyl-QS-18 2018 B) to             desarabinofuranosyl-QS-21 1856 component, such as:             -   desarabinofuranosyl-QS-18 2018 A component to                 desarabinofuranosyl-QS-21 1856 A component, such as:                 -   desarabinofuranosyl-QS-18 2018 A V1 to                     desarabinofuranosyl-QS-21 1856 A V1                 -   desarabinofuranosyl-QS-18 2018 A V2 to                     desarabinofuranosyl-QS-21 1856 A V2             -   desarabinofuranosyl-QS-18 2018 B component to                 desarabinofuranosyl-QS-21 1856 B component, such as:                 -   desarabinofuranosyl-QS-18 2018 B V1 to                     desarabinofuranosyl-QS-21 1856 B V1                 -   desarabinofuranosyl-QS-18 2018 B V2 to                     desarabinofuranosyl-QS-21 1856 B V2             -   desarabinofuranosyl-QS-18 2018 V1 component (i.e.                 desarabinofuranosyl-QS-18 2018 A V1 and/or                 desarabinofuranosyl-QS-18 2018 B V1) to                 desarabinofuranosyl-QS-21 1856 V1 component, such as:                 -   desarabinofuranosyl-QS-18 2018 A V1 to                     desarabinofuranosyl-QS-21 1856 A V1                 -   desarabinofuranosyl-QS-18 2018 B V1 to                     desarabinofuranosyl-QS-21 1856 B V1             -   desarabinofuranosyl-QS-18 2018 V2 component (i.e.                 desarabinofuranosyl-QS-18 2018 A V2 and/or                 desarabinofuranosyl-QS-18 2018 B V2) to                 desarabinofuranosyl-QS-21 1856 V2 component, such as:                 -   desarabinofuranosyl-QS-18 2018 A V2 to                     desarabinofuranosyl-QS-21 1856 A V2                 -   desarabinofuranosyl-QS-18 2018 B V2 to                     desarabinofuranosyl-QS-21 1856 B V2         -   desarabinofuranosyl-QS-18 1886 component (i.e.             desarabinofuranosyl-QS-18 1886 A and/or             desarabinofuranosyl-QS-18 1886 B) to             desarabinofuranosyl-QS-21 1712 component, such as:             -   desarabinofuranosyl-QS-18 1886 A component to                 desarabinofuranosyl-QS-21 1712 A             -   desarabinofuranosyl-QS-18 1886 B component to                 desarabinofuranosyl-QS-21 1712 B         -   desarabinofuranosyl-QS-18 2032 component (i.e.             desarabinofuranosyl-QS-18 2032 A and/or             desarabinofuranosyl-QS-18 2032 B) to             desarabinofuranosyl-QS-21 1870 component, such as:             -   desarabinofuranosyl-QS-18 2032 A component to                 desarabinofuranosyl-QS-21 1870 A component, such as:                 -   desarabinofuranosyl-QS-18 2032 A V1 to                     desarabinofuranosyl-QS-21 1870 A V1                 -   desarabinofuranosyl-QS-18 2032 A V2 to                     desarabinofuranosyl-QS-21 1870 A V2             -   desarabinofuranosyl-QS-18 2032 B component to QS-21,                 such as:                 -   desarabinofuranosyl-QS-18 2032 B V1 to                     desarabinofuranosyl-QS-21 1870 B V1                 -   QS-17 desarabinofuranosyl-QS-18 2032 B V2 to                     desarabinofuranosyl-QS-21 1870 B V2             -   desarabinofuranosyl-QS-18 2032 V1 component (i.e.                 desarabinofuranosyl-QS-18 2032 A V1 and/or                 desarabinofuranosyl-QS-18 2032 B V1) to                 desarabinofuranosyl-QS-21 1870 V1 component, such as:                 -   desarabinofuranosyl-QS-18 2032 A V1 to                     desarabinofuranosyl-QS-21 1870 A V1                 -   desarabinofuranosyl-QS-18 2032 B V1 to                     desarabinofuranosyl-QS-21 1870 B V1             -   desarabinofuranosyl-QS-18 2032 V2 component (i.e.                 desarabinofuranosyl-QS-18 2032 A V2 and/or                 desarabinofuranosyl-QS-18 2032 B V2) to                 desarabinofuranosyl-QS-21 1870 V2 component, such as:                 -   desarabinofuranosyl-QS-18 2032 A V2 to                     desarabinofuranosyl-QS-21 1870 A V2                 -   desarabinofuranosyl-QS-18 2032 B V2 to                     desarabinofuranosyl-QS-21 1870 B V2     -   acetylated desglucosyl-QS-17 components to acetylated QS-21         family components, such as:         -   acetylated desglucosyl-QS-17 2176 A to acetylated QS-21 2030             A, such as:             -   acetylated desglucosyl-QS-17 2176 A V1 to acetylated                 QS-21 2030 A V1             -   acetylated desglucosyl-QS-17 2176 A V2 to acetylated                 QS-21 2030 A V2         -   acetylated desglucosyl-QS-17 2044 A to acetylated QS-21 1898             A component         -   acetylated desglucosyl-QS-17 2190 A to acetylated QS-21 2044             A, such as:             -   acetylated desglucosyl-QS-17 2190 A V1 to acetylated                 QS-21 2044 A V1             -   acetylated desglucosyl-QS-17 2190 A V2 to acetylated                 QS-21 2044 A V2.

Enzymatic conversions may be applied to a single starting saponin or a plurality of starting saponins in parallel. It will be appreciated that a process may comprise or consist of the conversions specified above, depending on the composition of the starting material and the enzymes used. Furthermore, while a process may be limited to the use of a single enzyme intended to remove a particular sugar residue or group of sugar residues from (i) a single starting saponin, (ii) a family of starting saponins, or (iii) from a plurality of families of starting saponins; processes may also use a plurality of enzymes intended to remove a plurality of sugar residues from (i) a single starting saponin, (ii) a family of starting saponins, or (iii) from a plurality of families of starting saponins. Processes involving multiple enzymes may be undertaken in series (i.e. a single enzyme is applied to saponin material at any time) or in parallel (i.e. more than one enzyme is applied to saponin material at any time, such as two or three enzymes, in particular two enzymes), or combinations thereof.

Processes involving the removal of multiple sugar residues may involve the removal of single (but different) sugar residues from multiple starting saponins and/or the removal of multiple sugar residues from particular starting saponins (such as 2, 3 or 4 residues, in particular 2 or 3, especially 2 residues). Removal of multiple sugar residues from particular starting saponins may involve any combination of removal of single residues and/or removal of a plurality of residues in a single cleavage.

Exemplary processes may comprise (such as consist of) the removal of glucose and rhamnose, in particular an alpha-rhamnose residue and a beta-glucose residue, such as the alpha-L-rhamnose residue and the beta-D-glucose residue from quillaic acid glycosides:

Particular multi-sugar enzymatic conversions of interest include:

-   -   QS-17 family components to QS-21 family components, such as:         -   QS-17 2296 component (i.e. QS-17 2296 A and/or QS-17 2296 B)             to QS-21 1988 component, such as:             -   QS-17 2296 A component to QS-21 1988 A component, such                 as:                 -   QS-17 2296 A V1 to QS-21 1988 A V1                 -   QS-17 2296 A V2 to QS-21 1988 A V2             -   QS-17 2296 B component to QS-21 1988 B component, such                 as:                 -   QS-17 2296 B V1 to QS-21 1988 B V1                 -   QS-17 2296 B V2 to QS-21 1988 B V2             -   QS-17 2296 V1 component (i.e. QS-17 2296 A V1 and/or                 QS-17 2296 B V1) to QS-21 1988 V1 component, such as:                 -   QS-17 2296 A V1 to QS-21 1988 A V1                 -   QS-17 2296 B V1 to QS-21 1988 B V1             -   QS-17 2296 V2 component (i.e. QS-17 2296 A V2 and/or                 QS-17 2296 B V2) to QS-21 1988 V2 component, such as:                 -   QS-17 2296 A V2 to QS-21 1988 A V2                 -   QS-17 2296 B V2 to QS-21 1988 B V2         -   QS-17 2164 component (i.e. QS-17 2164 A and/or QS-17 2164 B)             to QS-21 1856 component, such as:             -   QS-17 2164 A component to QS-21 1856 A component             -   QS-17 2164 B component to QS-21 1856 B component         -   QS-17 2310 component (i.e. QS-17 2310 A and/or QS-17 2310 B)             to QS-21 2002 component, such as:             -   QS-17 2310 A component to QS-21 2002 A component, such                 as:                 -   QS-17 2310 A V1 to QS-21 2002 A V1                 -   QS-17 2310 A V2 to QS-21 2002 A V2             -   QS-17 2310 B component to QS-21 2002 B component, such                 as:                 -   QS-17 2310 B V1 to QS-21 2002 B V1                 -   QS-17 2310 B V2 to QS-21 2002 B V2             -   QS-17 2310 V1 component (i.e. QS-17 2310 A V1 and/or                 QS-17 2310 B V1) to QS-21 2002 V1, such as:                 -   QS-17 2310 A V1 to QS-21 2002 A V1                 -   QS-17 2310 B V1 to QS-21 2002 B V1             -   QS-17 2310 V2 component (i.e. QS-17 2310 A V2 and/or                 QS-17 2310 B V2) to QS-21 2002 V2, such as:                 -   QS-17 2310 A V2 to QS-21 2002 A V2                 -   QS-17 2310 B V2 to QS-21 2002 B V2.

Extracts may contain complex mixtures of saponin components and consequently may experience a plurality of conversions when multiple enzymes are present. For example, a starting mixture containing QS-17, QS-18 and desglucosyl-QS-17 components which is treated with an appropriate beta-glucosidase and alpha-rhamnosidase in parallel may undergo conversions including:

-   -   QS-18 family components to QS-21 family components, especially         QS-18 2150 component to QS-21 1988 component;     -   desglucosyl-QS-17 family components to QS-21 family components,         especially desglucosyl-QS-17 2134 component to QS-21 1988         component;     -   QS-17 family components to desglucosyl-QS-17 family components         to QS-21 components, especially QS-17 2296 component to         desglucosyl-QS-17 2134 component to QS-21 1988 component; and     -   QS-17 family components to QS-18 family components to QS-21         family components, especially QS-17 2296 component to QS-18 2150         component to QS-21 1988 component.

Enzyme Selection

Extensive protein or DNA databases of natural and artificial glycosidases are available. Candidate enzymes may be selected and screened to assess suitability for achieving a particular conversion under particular reaction conditions. Suitability of an enzyme will depend on a number of factors including:

-   -   target sugar (e.g. glucose, rhamnose)     -   target sugar anomer (alpha or beta);     -   target sugar enantiomer (D or L);     -   target sugar location (endo or exo); and     -   target sugar environment (e.g. chemical/physical, impacting         accessibility and reactivity).

Additional factors which facilitate effective conversions include:

-   -   rate of conversion;     -   environmental sensitivity—including pH, temperature, substrate,         product and contaminant concentration tolerance; and     -   specificity for target sugar, including in respect of other         sugar residues, other anomers, other sugar residue locations,         and between different residues of the same sugar anomer and         location within a substrate (if multiple such residues are         present).

Those skilled in the art will appreciate that the level and type of specificity required of an enzyme will depend on the objective to be achieved and the general circumstances.

Conversion of QS-18 family components to QS-21 family components requires an enzyme demonstrating beta exo glucosidase activity.

Conversion of QS-17 family components to desglucosyl-QS-17 family components requires an enzyme demonstrating beta exo glucosidase activity.

Conversion of desglucosyl-QS-17 family components to QS-21 family components requires an enzyme demonstrating alpha exo rhamnosidase activity.

Conversion of QS-17 family components to QS-18 family components requires an enzyme demonstrating alpha exo rhamnosidase activity.

It may be noted that many Quillaja saponaria starting saponins of interest contain only one glucose residue. Many Quillaja saponaria starting saponins of interest contain a plurality of rhamnose residues, therefore selectivity for specific rhamnose residues is of greater importance practically. For example, conversion of desglucosyl-QS-17 family components to QS-21 components or QS-17 family components to QS-18 family components requires specificity for exo-rhamnosidase action over endo-rhamnosidase action. Furthermore, rhamnosidase specificity for the alpha-O-rhamnosylation at the C2 position of the arabinofuranose moiety over other terminal rhamnose residues (e.g. in the rhamnose chemotype components) may also be desirable. In certain embodiments it may be desirable to remove the terminal rhamnose from rhamnose chemotype components (alone or in conjunction with any alpha-O-rhamnosylation at the C2 position of the arabinofuranose moiety), to better facilitate their chromatographic separation from xylose chemotype components.

In one embodiment saponin starting material is subjected to enzymatic modification by a single enzyme. The single enzyme may be a glucosidase, in particular a beta exo glucosidase. A single enzyme glucosidase may be an engineered glucosidase polypeptide of the present invention. Alternatively, the single enzyme is a rhamnosidase, in particular an alpha exo rhamnosidase. A single enzyme rhamnosidase may an engineered rhamnosidase polypeptide of the present invention.

Preferred enzymes are those which efficiently enzymatically convert a starting saponin(s) to the desired product saponin(s) while demonstrating limited or no undesired conversion(s) of other saponin components present.

In one embodiment saponin starting material is subjected to enzymatic modification by more than one enzyme, such as by two or three enzymes, especially by two enzymes. Enzymatic modification by more than one enzyme may involve sequential/series enzymatic modification. Alternatively, enzymatic modification by more than one enzyme may involve concurrent/parallel enzymatic modification. Enzymatic modification by at least three enzymes may involve a combination of sequential/series (modification by one enzyme) and concurrent/parallel (modification by at least two other enzymes) enzymatic modification, in any order. Where a plurality of enzymes are provided, these may be as distinct proteins or may be in the form of one or more fusion proteins.

An enzyme of interest is a glucosidase, such as a beta exo glucosidase. A glucosidase may be an engineered glucosidase polypeptide of the present invention. Another enzyme of interest is a rhamnosidase, such as an alpha exo rhamnosidase. A rhamnosidase may an engineered rhamnosidase polypeptide of the present invention. Enzyme combinations of interest include those comprising, such as consisting of, a glucosidase and a rhamnosidase, in particular a beta exo glucosidase and an alpha exo rhamnosidase. Enzymatic modification involving a glucosidase and a rhamnosidase, in particular a beta exo glucosidase and an alpha exo rhamnosidase, may be undertaken: sequentially with glucosidase (e.g. beta exo glucosidase) followed by rhamnosidase (e.g. alpha exo rhamnosidase), sequentially with rhamnosidase (e.g. alpha exo rhamnosidase) followed by glucosidase (e.g. beta exo glucosidase) or, conveniently, concurrently with both glucosidase (e.g. beta exo glucosidase) and rhamnosidase (e.g. alpha exo rhamnosidase). Particular enzyme combinations of interest are those comprising, such as consisting of, an engineered glucosidase of the present invention and an engineered rhamnosidase polypeptide of the present invention.

Enzymes utilised will typically be of external origin to saponin material i.e. not naturally found within the source of saponins obtained by extraction.

Enzymes may be native, i.e. naturally occurring glycosidases, or alternatively may be non-naturally occurring glycosidases. In one embodiment a glucosidase enzyme is a naturally occurring glucosidase (e.g. exo glucosidase, such as beta exo glucosidase). In a second embodiment a glucosidase enzyme is a non-naturally occurring glucosidase (e.g. exo glucosidase, such as beta exo glucosidase). In one embodiment a rhamnosidase enzyme is a naturally occurring rhamnosidase (e.g. exo rhamnosidase, such as alpha exo rhamnosidase). In a second embodiment a rhamnosidase enzyme is a non-naturally occurring rhamnosidase (e.g. exo rhamnosidase, such as alpha exo rhamnosidase).

Enzymes may be modified relative to a reference enzyme (‘engineered’). Point mutations, either singly or in combination, introduced by engineering may provide benefits such as increased activity, increased specificity, increased stability, increased expression or other the like. Assays to confirm the properties of the enzymes are well known to those skilled in the field. For example, activity may be quantified by methods such as those shown in the examples (see Examples 4 to 7) or by analogous methods.

Different enzymes may show different sensitivity to environmental conditions, such as pH, temperature, substrate concentration, product concentration, solvent composition, presence of contaminants and the like. Such parameters may be taken into consideration during screening of candidate enzymes for the desired activity.

Candidate enzymes having beta glucosidase activity include those in EC3.2.1.21.

Beta exo glucosidases of interest include those described in Table 7, especially SEQ ID Nos. 262, 208, 63, 229, 250, 5, 101, 207, 169, 247, 302, 324, 319, 9, 240, 325 and 338, and functional variants thereof. Particular beta exo glucosidases of interest include SEQ ID Nos. 262, 208, 63, 229, 250, 5, 101, 207, 169, 247, 302, 324 and 319, and functional variants thereof, such as SEQ ID Nos. 262, 208, 63, 229, 250, 5, 101 and 207, and functional variants thereof.

Another group of beta exo glucosidases of interest include those described in Table 9, especially SEQ ID Nos. 850, 879, 868, 826, 804, 888, 881, 891, 816, 827, 857, 853, 842, 814, 886, 885, 838, 829, 808, 828, 870, 873, 844, 882, 874, 825, 824, 823, 810, 894, 849, 803, 890, 841, 832, 830, 845, 871, 837, 883 and 809, and functional variants thereof. Particular beta exo glucosidases of interest include SEQ ID Nos. 850, 879, 868, 826, 804, 888, 881, 891, 816, 827, 857, 853, 842, 814, 886, 885, 838, 829, 808, 828, 870, 873, 844, 882, 874, 825, 824, 823, 810, 894, 849, 803, 890 and 841, and functional variants thereof, such as SEQ ID Nos. 850, 879, 868, 826, 804, 888, 881, 891, 816, 827, 857, 853, 842, 814, 886, 885, 838, 829, 808, 828, 870, 873, 844, 882, 874, 825, 824, 823, 810 and 894, and functional variants thereof.

SEQ ID No. 262, and functional variants thereof, are particularly desirable beta exo glucosidases. In one embodiment the beta exo glucosidase comprises, such as consists of: (i) SEQ ID. 262; or (ii) a functional variant thereof having at least 80% identity to SEQ ID. 262, especially at least 90%, in particular at least 95%, such as at least 96%, at least 97%, at least 98%, for example at least 99% identity; or (iii) a functional fragment of at least 100, especially at least 200, particularly at least 300, such as at least 400, for example at least 500 contiguous amino acids of SEQ ID. 262.

Candidate enzymes having alpha rhamnosidase activity include those in EC3.2.1.40.

Alpha exo rhamnosidases of interest include SEQ ID Nos. 992, 1003, 1052, 1073, 1017, 1055, 1075, 1001, 1007, 1061, 1079, 1027, 1039, 1041, 989, 1053, 1018, 1066, 1082, 1076, 993, 1077, 1046, 1015, 1063, 1054, 1074, 1067 and 1033, and functional variants thereof. Particular alpha exo rhamnosidases of interest include SEQ ID Nos. 992, 1003, 1052, 1073, 1017, 1055, 1075, 1001, 1007, 1061, 1079, 1027, 1039, 1041, 989, 1053, 1018, 1066, 1082, 1076, 993 and 1077, and functional variants thereof, such as SEQ ID Nos. 992, 1003, 1052, 1073, 1017, 1055, 1075, 1001, 1007, 1061, 1079, 1027, 1039, 1041 and 989, and functional variants thereof.

SEQ ID No. 1017, and functional variants thereof, are particularly desirable exo rhamnosidases. In one embodiment the alpha exo rhamnosidase comprises, such as consists of: (i) SEQ ID. 1017; or (ii) a functional variant thereof having at least 80% identity to SEQ ID. 1017, especially at least 90%, in particular at least 95%, such as at least 96%, at least 97%, at least 98%, for example at least 99% identity; or (iii) a functional fragment of at least 100, especially at least 200, particularly at least 300, such as at least 400, for example at least 500 contiguous amino acids of SEQ ID. 1017.

Functional variants of interest in the present application include those comprising, such as consisting of: (i) a sequence having at least 80% identity to the reference sequence, especially at least 90%, in particular at least 95%, such as at least 96%, at least 97%, at least 98%, for example at least 99% identity; or (ii) a fragment of at least 100, especially at least 200, particularly at least 300, such as at least 400, for example at least 500 contiguous amino acids of the reference sequence.

Certain desirable functional variants of interest include those comprising, such as consisting of, a sequence having 1 to 20 additions, deletions and/or substitutions relative to the reference sequence, especially 1 to 15 additions, deletions and/or substitutions, particularly 1 to additions, deletions and/or substitutions, such as 1 to 5 additions, deletions and/or substitutions.

The degree of sequence identity may be determined using by the homology alignment algorithm of Needleman and Wunsch, the ClustalW program or the BLASTP algorithm, using default settings. An algorithm using global alignment (Needleman and Wunsch) is preferred.

“Percentage of sequence identity,” “percent identity,” and “percent identical” are used herein to refer to comparisons between polynucleotide sequences or polypeptide sequences, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Determination of optimal alignment and percent sequence identity is performed using the BLAST and BLAST 2.0 algorithms (see, e.g., Altschul, 1990; Altschul, 1997). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. Briefly, the BLAST analyses involve first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (N) of 11, an expectation (E) of 10, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff, 1989).

Numerous other algorithms are available that function similarly to BLAST in providing percent identity for two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith, 1981, by the homology alignment algorithm of Needleman, 1970, by the search for similarity method of Pearson, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology, 1995)). Additionally, determination of sequence alignment and percent sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided. The ClustalW program is also suitable for determining identity.

Modestobacter marinus glucosidase (Uniparc reference UPI000260A2FA, Uniprot reference I4EYD5-SEQ ID No. 262 herein) is a naturally occurring glucosidase demonstrating beta exo glucosidase activity and, for example, is capable of the conversion of QS-18 family components to QS-21 family components. Despite its potent activity, the present inventors have found that the properties of wild type Modestobacter marinus glucosidase may be altered by the introduction of one or more mutations.

The present invention provides an engineered glucosidase polypeptide comprising, such as consisting of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID No. 262, or a functional fragment thereof, wherein the engineered glucosidase polypeptide includes at least one residue substitution from:

-   -   F44Y;     -   V60L;     -   G117A;     -   F170N;     -   V263G or V263L;     -   N351H or N351Q;     -   A355H, A355I, A355L, A355M, A355R, A355T or A355W;     -   A356P;     -   R357A, R357C, R357K, R357M or R357Q;     -   G362C;     -   T365A, T365N or T365S;     -   L367C;     -   V394R;     -   V395Y;     -   Q396E, Q396G, Q396N, Q396P, Q396R, Q396S or Q396Y;     -   F430W;     -   R435F;     -   V438T;     -   V440F;     -   F442M or F442Q;     -   G444T;     -   A473F or A473R;     -   L474C, L474I or L474V;     -   I475F;     -   L492C, L492G, L492H, L492I, L492N, L492Q, L492V, L492W or L492Y;     -   Q493F or Q493H;     -   P494H or P494I;     -   S495I, S495K or S495Q;     -   G496P or G496W;     -   D498A, D498E, D498F, D498I, D498K, D498L, D498N, D498P, D498R,         D498S, D498T or D498V;     -   A502R;     -   M504G or M504R;     -   L507A or L507R;     -   T508M;     -   L529M;     -   F535P;     -   A536D or A536E;     -   A537R;     -   F541A, F541I, F541L, F541M or F541V;     -   L542I;     -   Q543G or Q543L;     -   E547L; and     -   Y585W.

The glucosidases will contain one to forty-two of the substitutions, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six to thirty or thirty-one to forty-three substitutions.

The present invention also provides an engineered glucosidase polypeptide comprising, such as consisting of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID No. 262, or a functional fragment thereof, wherein the engineered glucosidase polypeptide includes at least one residue substitution from:

-   -   F44Y;     -   V263L;     -   N351H;     -   A355H, A355M or A355W;     -   R357M;     -   T365N;     -   L367C;     -   Q396R;     -   V438T;     -   F442Q;     -   L474C;     -   I475F;     -   L492V, L492N or L492H,     -   M504R;     -   L507R; and     -   F541I.

The glucosidases will contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or all sixteen substitutions.

The engineered glucosidase polypeptide may comprise, such as consist of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID No. 262, or a functional fragment thereof, wherein the engineered glucosidase polypeptide includes at least one residue substitution from: F44Y, V263L, A355W, R357M, T365N, L367C, Q396R, F442Q, L474C, I475F and F541I.

Suitably the engineered glucosidase polypeptide comprises, such as consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID No. 262, or a functional fragment thereof, wherein the engineered glucosidase polypeptide includes the residue substitutions: F44Y, V263L, A355W, R357M, T365N, L367C, Q396R, F442Q, L474C, I475F and F541I.

The present invention provides a polypeptide comprising an amino acid sequence of sequence of SEQ ID No. 262 with one to twenty-five mutations selected from the list consisting of:

-   -   (i) F44Y     -   (ii) V263L     -   (iii) N351H     -   (iv) A355H, A355I, A355L, A355M, A355R, A355T or A355W     -   (v) A356P     -   (vi) R357M     -   (vii) T365N     -   (viii) L3670     -   (ix) F442Q     -   (x) G443D     -   (xi) A473F     -   (xii) L474O     -   (xiii) I475F     -   (xiv) L492H, L492N, L492V     -   (xv) P494I     -   (xvi) G496P     -   (xvii) D498P     -   (xviii) M504R     -   (xix) L507R     -   (xx) F535P     -   (xxi) A537R     -   (xxii) F541I     -   (xxiii) L542I;     -   (xxiv) E547L and     -   (xxv) E588K.

Variant glucosidases will contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four or all twenty-five mutations.

In some embodiments an engineered glucosidase is not a polypeptide comprising an amino acid sequence of sequence of SEQ ID No. 262 with one to twenty-five mutations selected from the list consisting of:

-   -   (i) F44Y     -   (ii) V263L     -   (iii) N351H     -   (iv) A355H, A355I, A355L, A355M, A355R, A355T or A355W     -   (v) A356P     -   (vi) R357M     -   (vii) T365N     -   (viii) L367C     -   (ix) F442Q     -   (x) G443D     -   (xi) A473F     -   (xii) L474C     -   (xiii) I475F     -   (xiv) L492H, L492N, L492V     -   (xv) P494I     -   (xvi) G496P     -   (xvii) D498P     -   (xviii) M504R     -   (xix) L507R     -   (xx) F535P     -   (xxi) A537R     -   (xxii) F541I     -   (xxiii) L542I     -   (xxiv) E547L and     -   (xxv) E588K.

The above-mentioned engineered glucosidase polypeptides may also be referred to herein as examples of ‘variant glucosidases’.

A variant glucosidase may contain F44Y.

A variant glucosidase may contain V60L.

A variant glucosidase may contain G117A.

A variant glucosidase may contain F170N.

A variant glucosidase may contain V263G or V263L, in particular V263L.

A variant glucosidase may contain N351H or N351Q, in particular N351H.

A variant glucosidase may contain A355H, A355I, A355L, A355M, A355R, A355T or A355W. In some embodiments a variant glucosidase contains A355H. In some embodiments a variant glucosidase contains A355I. In some embodiments a variant glucosidase contains A355L. In some embodiments a variant glucosidase contains A355M. In some embodiments a variant glucosidase contains A355R. In some embodiments a variant glucosidase contains A355T. In some embodiments a variant glucosidase contains A355W.

A variant glucosidase may contain A356P.

A variant glucosidase may contain R357A, R357C, R357K, R357M or R357Q, in particular R357M.

A variant glucosidase may contain G362C.

A variant glucosidase may contain T365A, T365N or T365S, in particular T365N.

A variant glucosidase may contain L367C.

A variant glucosidase may contain V394R.

A variant glucosidase may contain V395Y.

A variant glucosidase may contain Q396E, Q396G, Q396N, Q396P, Q396R, Q396S or Q396Y, in particular Q396R.

A variant glucosidase may contain F430W.

A variant glucosidase may contain R435F.

A variant glucosidase may contain V438T.

A variant glucosidase may contain V440F.

A variant glucosidase may contain F442M or F442Q, in particular F442Q.

A variant glucosidase may contain G443D.

A variant glucosidase may contain G444T.

A variant glucosidase may contain A473F or A473R, in particular A473F.

A variant glucosidase may contain L474O, L474I or L474V, in particular L474C.

A variant glucosidase may contain I475F.

A variant glucosidase may contain L4920, L492G, L492H, L492I, L492N, L492Q, L492V, L492W or L492Y, in particular L492H, L492N, L492V. In some embodiments a variant glucosidase contains L492H. In some embodiments a variant glucosidase contains L492N. In some embodiments a variant glucosidase contains L492V.

A variant glucosidase may contain Q493F or Q493H.

A variant glucosidase may contain P494H or P494I, in particular P494I.

A variant glucosidase may contain S495I, S495K or S495Q.

A variant glucosidase may contain G496P or G496W, in particular G496P.

A variant glucosidase may contain D498A, D498E, D498F, D498I, D498K, D498L, D498N, D498P, D498R, D498S, D498T or D498V, in particular D498P.

A variant glucosidase may contain A502R.

A variant glucosidase may contain M504G or M504R, in particular M504R.

A variant glucosidase may contain L507A or L507R, in particular L507R.

A variant glucosidase may contain T508M.

A variant glucosidase may contain L529M.

A variant glucosidase may contain F535P.

A variant glucosidase may contain A536D or A536E.

A variant glucosidase may contain A537R.

A variant glucosidase may contain F541A, F541I, F541L, F541M or F541V, in particular F541I.

A variant glucosidase may contain L542I.

A variant glucosidase may contain Q543G or Q543L.

A variant glucosidase may contain E547L.

A variant glucosidase may contain Y585W.

A variant glucosidase may contain E588K.

Variant glucosidases may comprise R357M, T365N, A473F, L474O and I475F.

Variant glucosidases may comprise F44Y, R357M, T365N, F442Q, A473F, L474O and I475F.

Variant glucosidases may comprise F44Y, V263L, R357M, T365N, F442Q, A473F, L474C, I475F and F541I.

Variant glucosidases may comprise F44Y, V263L, A355W, R357M, T365N, L367C, Q396R, F442Q, L474O, I475F and F541I.

Variant glucosidases may comprise F44Y, V263L, R357M, T365N, F442Q, L474O, I475F, F541I and zero to seventeen mutations selected from the list consisting of:

-   -   (iii) N351H     -   (iv) A355H, A355I, A355L, A355M, A355R, A355T or A355W     -   (v) A356P     -   (viii) L367C     -   (x) G443D     -   (xi) A473F     -   (xiv) L492H, L492N, L492V     -   (xv) P494I     -   (xvi) G496P     -   (xvii) D498P     -   (xviii) M504R     -   (xix) L507R     -   (xx) F535P     -   (xxi) A537R     -   (xxiii) L542I     -   (xxiv) E547L and     -   (xxv) E588K.

A variant glucosidase may comprise a “tag,” a sequence of amino acids that allows for the isolation and/or identification of the polypeptide. For example, adding an affinity tag can be useful in purification. Exemplary affinity tags that can be used include histidine (HIS) tags (e.g., hexa histidine-tag, or 6×His-Tag), FLAG-TAG, and HA tags. Tags may be located N-terminally or C-terminally and may be directly connected or attached via a linking sequence. SEQ ID No. 1177 provides a sequence for an exemplary 6×His-Tag with linker sequence which may be N-terminally attached. SEQ ID No. 1178 provides a sequence for an exemplary 6×His-Tag with linker sequence which may be C-terminally attached.

In certain embodiments, the tags used herein are removable, e.g., removal by chemical agents or by enzymatic means, once they are no longer needed, e.g., after the polypeptide has been purified.

A variant glucosidase may comprise 1000 residues or fewer, especially 950 residues or fewer, in particular 900 residues or fewer, such as 850 residues or fewer.

A variant glucosidase may consist of an amino acid sequence of SEQ ID No. 262 with one to twenty-five mutations selected from the list consisting of:

-   -   (i) F44Y     -   (ii) V263L     -   (iii) N351H     -   (iv) A355H, A355I, A355L, A355M, A355R, A355T or A355W     -   (v) A356P     -   (vi) R357M     -   (vii) T365N     -   (viii) L367C     -   (ix) F442Q     -   (x) G443D     -   (xi) A473F     -   (xii) L474C     -   (xiii) I475F     -   (xiv) L492H, L492N, L492V     -   (xv) P494I     -   (xvi) G496P     -   (xvii) D498P     -   (xviii) M504R     -   (xix) L507R     -   (xx) F535P     -   (xxi) A537R     -   (xxii) F541I     -   (xxiii) L542I     -   (xxiv) E547L and     -   (xxv) E588K.

Variant glucosidases desirably demonstrate a FIOP (Fold Improvement Over Parent) relative to SEQ ID No. 262 of at least 1.05, especially at least 2, in particular at least 10, such as at least 50. FIOP may be determined by the methods described in Example 4.

Kribbella flavida rhamnosidase (Uniparc reference UPI00019BDB13, Uniprot reference D2PMT5—SEQ ID No. 1017 herein) is a naturally occurring rhamnosidase demonstrating alpha exo rhamnosidase activity and, for example, is capable of the conversion of desglucosyl-QS-17 family components to QS-21 family components. Despite its potent activity, the present inventors have found that the properties of wild type Kribbella flavida rhamnosidase may be altered by the introduction of one or more mutations.

The present invention provides an engineered rhamnosidase polypeptide comprising, such as consisting of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID No. 1017, or a functional fragment thereof, wherein the engineered rhamnosidase polypeptide includes at least one residue substitution from:

-   -   (i) A56C     -   (ii) A143P     -   (iii) Q181H, Q181R or Q181S     -   (v) G215S     -   (vi) F216M     -   (vii) G218D or G218N     -   (viii) K219G     -   (ix) A238M     -   (x) T252Y     -   (xi) T311W     -   (xii) V326C     -   (xiii) G357C     -   (xiv) S369C, S369I, S369K or S369M     -   (xv) I487M, I487Q or I487V     -   (xvi) K492N     -   (xvii) V499T     -   (xviii) G508S     -   (xix) R543C     -   (xx) L557Y     -   (xxi) G634A     -   (xxii) S635N     -   (xxiii) A690C and     -   (xxiv) Q921H.

Consequently, the present invention provides a polypeptide comprising an amino acid sequence of sequence of SEQ ID No. 1017 with one to twenty-four mutations selected from the list consisting of:

-   -   (i) A56C     -   (ii) A143P     -   (iii) Q181H, Q181R or Q181S     -   (iv) L214M     -   (v) G215S     -   (vi) F216M     -   (vii) G218D or G218N     -   (viii) K219G     -   (ix) A238M     -   (x) T252Y     -   (xi) T311W     -   (xii) V326C     -   (xiii) G357C     -   (xiv) S369C, S369I, S369K or S369M     -   (xv) I487M, I487Q or I487V     -   (xvi) K492N     -   (xvii) V499T     -   (xviii) G508S     -   (xix) R543C     -   (xx) L557Y     -   (xxi) G634A     -   (xxii) S635N     -   (xxiii) A690C and     -   (xxiv) Q921H.

Such polypeptides may be referred to herein as ‘variant rhamnosidases’.

Variant rhamnosidases will contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three or all twenty-four mutations.

A variant rhamnosidase may contain A56C.

A variant rhamnosidase may contain A143P.

A variant rhamnosidase may contain Q181H, Q181R or Q181S. In some embodiments a variant rhamnosidase contains Q181H. In some embodiments a variant rhamnosidase contains Q181R. In some embodiments a variant rhamnosidase contains Q181S.

A variant rhamnosidase may contain L214M.

A variant rhamnosidase may contain G215S.

A variant rhamnosidase may contain F216M.

A variant rhamnosidase may contain G218D or G218N. In some embodiments a variant rhamnosidase contains G218D. In some embodiments a variant rhamnosidase contains G218N.

A variant rhamnosidase may contain K219G.

A variant rhamnosidase may contain A238M.

A variant rhamnosidase may contain T252Y.

A variant rhamnosidase may contain T311W.

A variant rhamnosidase may contain V326C.

A variant rhamnosidase may contain G357C.

A variant rhamnosidase may contain S369C, S369I, S369K or S369M. In some embodiments a variant rhamnosidase contains S369C. In some embodiments a variant rhamnosidase contains S369I. In some embodiments a variant rhamnosidase contains S369K. In some embodiments a variant rhamnosidase contains S369M.

A variant rhamnosidase may contain I487M, I487Q or I487V. In some embodiments a variant rhamnosidase contains I487M. In some embodiments a variant rhamnosidase contains I487Q. In some embodiments a variant rhamnosidase contains I487V.

A variant rhamnosidase may contain K492N.

A variant rhamnosidase may contain V499T.

A variant rhamnosidase may contain G508S.

A variant rhamnosidase may contain R543C.

A variant rhamnosidase may contain L557Y.

A variant rhamnosidase may contain G634A.

A variant rhamnosidase may contain S635N.

A variant rhamnosidase may contain A690C.

A variant rhamnosidase may contain Q921H.

Variant rhamnosidases may comprise A143P, L214M, K219G and Q921H.

Variant rhamnosidases may comprise A143P, L214M, K219G, G357C and Q921H.

Variant rhamnosidases may comprise A143P, L214M, G215S, G218N, K219G, G357C, G508S, G634A and Q921H.

Variant rhamnosidases may comprise A143P, L214M, G215S, G218D, K219G, G357C, G508S, G634A, A690C and Q921H.

Variant rhamnosidases may comprise A143P, L214M, G215S, K219G, G357C, G508S, G634A and Q921H and one to sixteen mutations selected from the list consisting of:

-   -   (i) A56C     -   (iii) Q181H, Q181R or Q181S     -   (vi) F216M     -   (vii) G218D or G218N     -   (ix) A238M     -   (x) T252Y     -   (xi) T311W     -   (xii) V326C     -   (xiv) S369C, S369I, S369K or S369M     -   (xv) I487M, I487Q or I487V     -   (xvi) K492N     -   (xvii) V499T     -   (xix) R543C     -   (xx) L557Y     -   (xxii) S635N and     -   (xxiii) A690C.

Variant rhamnosidases may comprise A143P, L214M, G215S, K219G, G357C, G508S, G634A, Q921H, G218D or G218N, and one to fifteen mutations selected from the list consisting of:

-   -   (i) A56C     -   (iii) Q181H, Q181R or Q181S     -   (vi) F216M     -   (ix) A238M     -   (x) T252Y     -   (xi) T311W     -   (xii) V326C     -   (xiv) S369C, S369I, S369K or S369M     -   (xv) I487M, I487Q or I487V     -   (xvi) K492N     -   (xvii) V499T     -   (xix) R543C     -   (xx) L557Y     -   (xxii) S635N and     -   (xxiii) A690C.

A variant rhamnosidase may comprise a “tag,” a sequence of amino acids that allows for the isolation and/or identification of the polypeptide. For example, adding an affinity tag can be useful in purification. Exemplary affinity tags that can be used include histidine (HIS) tags (e.g., hexa histidine-tag, or 6×His-Tag), FLAG-TAG, and HA tags. Tags may be located N-terminally or C-terminally and may be directly connected or attached via a linking sequence. SEQ ID No. 1177 provides a sequence for an exemplary 6×His-Tag with linker sequence which may be N-terminally attached. SEQ ID No. 1178 provides a sequence for an exemplary 6×His-Tag with linker sequence which may be C-terminally attached.

In certain embodiments, the tags used herein are removable, e.g., removal by chemical agents or by enzymatic means, once they are no longer needed, e.g., after the polypeptide has been purified.

A variant rhamnosidase may comprise 1100 residues or fewer, especially 1050 residues or fewer, in particular 1000 residues or fewer, such as 950 residues or fewer.

A variant rhamnosidase may consist of an amino acid sequence of SEQ ID No. 1017 with one to twenty-four mutations selected from the list consisting of:

-   -   (i) A56C     -   (ii) A143P     -   (iii) Q181H, Q181R or Q181S     -   (iv) L214M     -   (v) G215S     -   (vi) F216M     -   (vii) G218D or G218N     -   (viii) K219G     -   (ix) A238M     -   (x) T252Y     -   (xi) T311W     -   (xii) V326C     -   (xiii) G357C     -   (xiv) S369C, S369I, S369K or S369M     -   (xv) I487M, I487Q or I487V     -   (xvi) K492N     -   (xvii) V499T     -   (xviii) G508S     -   (xix) R543C     -   (xx) L557Y     -   (xxi) G634A     -   (xxii) S635N     -   (xxiii) A690C and     -   (xxiv) Q921H.

Variant rhamnosidases desirably demonstrate a FIOP relative to SEQ ID No. 1017 of at least 1.05, especially at least 2, in particular at least 10, such as at least 50. FIOP may be determined by the methods described in Example 4.

Function, in respect of functional variants, requires that the glycosidase activity is not notably reduced as a result of sequence variation, typically at least 50% of glycosidase activity, especially at least 75% activity, particularly at least 90%, such as at least 100% activity is maintained for at least one saponin modification reaction under at least one set of conditions (activity being determined by rate of modification of starting saponin to product saponin). Variants may be created with the intention of improving the glycosidase in some manner (e.g. conversion rate; specificity, which may be increased or reduced depending on needs; tolerance to environmental conditions, such as pH, substrate concentration, product concentration, other contaminants and the like; stability, thermal or chemical; production, such as facilitating expression or purification of the glycosidase either pre- or post-saponin modification). Variants need not be improved in all respects and may simply demonstrate a different balance of characteristics relative to the reference sequence.

Glycosidases will typically be 2000 amino acids or fewer, such as 1500 amino acids or fewer.

Suitably, glycosidases are soluble.

Glycosidases may be immobilised, such as by attachment to solid (e.g. polymer) particles. Immobilisation of glycosidases may facilitate separation from a reaction mixture, improve thermal stability and/or tolerance to environmental conditions.

Glycosidases may comprise a “tag,” a sequence of amino acids that allows for the isolation and/or identification of the polypeptide. For example, adding an affinity tag can be useful in purification. Exemplary affinity tags that can be used include histidine (HIS) tags (e.g., hexa histidine-tag, or 6×His-Tag), FLAG-TAG, and HA tags. Tags may be located N-terminally or C-terminally and may be directly connected or attached via a linking sequence. SEQ ID No. 1177 provides a sequence for an exemplary 6×His-Tag with linker sequence which may be N-terminally attached. SEQ ID No. 1178 provides a sequence for an exemplary 6×His-Tag with linker sequence which may be C-terminally attached.

Reaction Conditions

Any suitable reaction conditions may be used. Optimal conditions will depend on a range of factors including the identity of the starting saponin, product saponin, enzyme utilised and the like.

The reaction requires treatment of a starting saponin(s) with a glycosidase(s). Appropriate glycosidases may be added to a saponin containing composition in a range of forms such as solution (typically aqueous), suspension (typically aqueous) or solid. Glycosidases may be in a purified, partially purified (such as clarified cell lysate) or unpurified form (crude cell lysate or unlysed cells). The use of partially purified or unpurified forms may be of interest when source cells (e.g. recombinant host cells, such as E. coli) express the enzyme to an extent that desired activity sufficiently exceeds any deleterious impact arising from other host cell contaminants. Desirably the glycosidase(s) are added in the form of clarified lysates. Glycosidases may be freshly prepared (e.g. clarified lysate) or taken from storage, such as thawed frozen liquid (e.g. clarified lysate) or reconstituted dried material (e.g. freeze-dried clarified lysate). Where a plurality of glycosidases is used in parallel, these will typically be expressed in different host cells to ensure adequate process control. A plurality of glycosidases used in parallel may be added together or separately (in the same or different forms).

Glycosidases may be produced using a protein secretion system, such as Bacillus licheniformis.

The weight of a glycosidase present may be in the range of 0.0001 mg to 25 mg per ml, especially 0.0001 mg to 5 mg per ml, in particular 0.0001 mg to 1 mg per ml, such as 0.001 mg to 0.5 mg per ml. When provided in the form of dried clarified lysate, the weight of a glycosidase present may be in the range of 0.01 mg to 100 mg of lysate per ml, especially 0.01 mg to 30 mg per ml, in particular 0.01 mg to 5 mg per ml, such as 0.01 mg to 1 mg per ml.

Any appropriate pH may be used, though typically between pH 4 to 9, especially pH 5 to 8, and in particular pH 5.5 to 7.5 such as pH 5.5 to 6.5. Where a plurality of glycosidases is used in series, each enzymatic modification may be undertaken at a different pH though for convenience they may be undertaken at the same pH.

Buffers may be used to aid control of the pH. Suitable buffers and appropriate concentrations may be obtained from standard sources. Inorganic salt buffers are typically used, such as potassium phosphate, sodium phosphate, potassium acetate, sodium acetate, potassium citrate, sodium citrate and the like. A suitable buffer concentration may be 10 mM to 500 mM, especially 25 mM to 250 mM and in particular 50 mM to 100 mM. Buffer concentrations of about 50 mM, such as 50 mM or about 100 mM, such as 100 mM, may be used.

Any appropriate temperature may be used, though typically between 10 degC to 60 degC, especially 15 degC to 50 degC, in particular 15 degC to 45 degC, such as 20 degC to 42 degC.

An appropriate time such that the reaction proceeds sufficiently is usually up to 10 days, especially up to 5 days, in particular up to 3 days. Desirably the enzyme and reaction conditions are chosen such that the reaction proceeds sufficiently in a period of up to 2 days, especially up to 1 day, in particular up to 18 hrs, such as 12 hrs, for example up to 6 hrs.

The reaction will be undertaken in a suitable solvent, typically water or an aqueous solution with water miscible co-solvent(s) such as methanol, ethanol, n-propanol, i-propanol, tetrahydrofuran, ethylene glycol, glycerol,1,3-propanediol or acetonitrile. Any co-solvent(s) should be present in amounts which are not excessively deleterious to the reaction proceeding, such as 50% or less v/v, especially 20% or less, in particular 10% or less, such as 5% or less, for example 2% or less (in total).

The reaction may be homogeneous or heterogeneous, monophasic, bi-phasic or multiphasic with particulates, dispersed solids in suspension and/or colloidal micelles present. Desirably the reaction will be monophasic.

The starting saponins may be present at a concentration of 0.001 to 100 g per litre, especially 0.005 to 75 g per litre, in particular 0.01 to 50 g per litre, such as 0.1 to 25 g per litre, for example 1 to 10 g per litre.

The reaction may be carried out in various modes of operation such as batch mode, fed batch mode or continuous mode.

The reaction is typically performed at a scale which can provide commercial quantities of product saponin. A batch reaction volume may be at least 10 ml, especially at least 100 ml, in particular at least 1 L. A batch reaction volume may be 500 ml to 2000 L, especially 1 L to 1000 L, in particular 10 L to 500 L, such as 25 L to 200 L.

Completion and Mass Balance

Enzymes are desirably adequately selective for the conversion of a starting saponin into a product saponin rather than other conversions of the starting saponin. As used herein, the term selectivity means at least 25% (mole basis) of converted starting saponin results in the intended product saponin, in particular at least 50%, especially at least 75%, such as at least 90% (e.g. at least 95%).

The concept of selectivity may also be applied in the context of the conversion of a plurality of starting saponins into a plurality of product saponins such that at least 25% of converted starting saponins (mole basis) result in the intended product saponins, in particular at least 50%, especially at least 75%, such as at least 90% (e.g. at least 95%).

Desirably conversion of a starting saponin into a product saponin is complete. However, rate of conversion, specificity of conversion (including rate of non-specific conversion(s)), product inhibition, starting saponin stability under reaction conditions, product saponin stability under reaction conditions and the like mean that conversions may not be complete or that it is desirable (e.g. for maximum yield or to obtain a balance between yield and process time) for a conversion to be stopped prior to completion.

Removal of Enzymes

At the point the reaction has progressed to the desired extent it may be stopped by denaturing or otherwise removing the enzyme. For example, the pH of reaction mixture may be adjusted to about pH to 3.5 to 4, especially pH 3.5 to 4, in particular pH 3.8 and/or the addition of sufficient quantities of anti-solvents or denaturing solvents such as acetonitrile. Precipitated enzyme may be removed by filtration.

Other Definitions

By the term ‘Preceding peak’ is meant the peak immediately preceding the QS-21 main peak in the HPLC-UV methods described herein (see FIG. 2 ).

By the term ‘m/z’ is meant the mass to charge ratio of the monoisotope peak. Unless otherwise specified, ‘m/z’ is determined by negative ion electrospray mass spectrometry.

By the term ‘ion abundance’ is meant the amount of a specified m/z measured in the sample, or in a given peak as required by the context. The mass chromatogram for the specified m/z may be extracted from the MS total ion chromatogram in the UPLC-UV/MS methods described herein. The mass chromatogram plots the signal intensity versus time. Ion abundance is measured as the area of the integrated peak. The area for a specified m/z/area for a relative reference m/z=relative abundance.

By the term ‘UV absorbance at 214 nm’ is meant the area of an integrated peak in the UV absorbance chromatogram. The (area for a specified peak)/(area of all integrated peaks in the chromatogram)×100=percentage area for the specified peak.

By the term ‘UV absorbance at 214 nm and relative ion abundance’ is meant an estimate for the percentage of a given m/z for co-eluting species. (Percentage area for given UV peak)×(relative ion abundance for m/z of interest in given peak)/(sum of all relative ion abundance for given peak)=percentage of m/z of interest in the given UV peak, assumes relative ion abundance included for all coeluting species.

By the term ‘wherein the monoisotope of the most abundant species is 1988 m/z’ is meant the monoisotope of the most abundant species, first peak in the isotopic group with highest response per m/z is m/z 1987.9. The most abundant species may be determined by creating a combined spectrum across the entire total ion chromatogram using the UPLC-UV/MS method (negative ion electrospray) as described herein.

By the term ‘dried’ is meant that substantially all solvent has been removed. A dried extract will typically contain less than 5% solvent w/w, especially less than 2.5% (such as less than 5% water w/w, especially less than 2.5%). Suitably the dried extract will contain 100 ppm or less acetonitrile (w/w).

Further, there is provided a method for the manufacture of a saponin composition comprising the steps of:

-   -   (i) treating a crude aqueous extract of Quillaja saponaria by         polyvinylpolypyrrolidone (PVPP) adsorption;     -   (ii) enzymatically modifying the treated extract with a         glucosidase and/or a rhamnosidase;     -   (iii) purifying the modified extract by reverse phase         chromatography using a polystyrene resin; and     -   (iv) purifying the modified extract by reverse phase         chromatography using a phenyl resin to provide a saponin         composition.

There is also provided a method for the manufacture of a saponin composition comprising the steps of:

-   -   (i) treating a crude aqueous extract of Quillaja saponaria by         polyvinylpolypyrrolidone adsorption;     -   (ii) enzymatically modifying the treated extract with an         engineered glucosidase polypeptide;     -   (iii) purifying the modified extract by reverse phase         chromatography using a polystyrene resin; and     -   (iv) purifying the modified extract by reverse phase         chromatography using a phenyl resin to provide a saponin         composition.

Additionally provided a method for the manufacture of a saponin composition comprising the steps of:

-   -   (i) treating a crude aqueous extract of Quillaja saponaria by         polyvinylpolypyrrolidone adsorption;     -   (ii) enzymatically modifying the treated extract with an         engineered rhamnosidase polypeptide;     -   (iii) purifying the modified extract by reverse phase         chromatography using a polystyrene resin; and     -   (iv) purifying the modified extract by reverse phase         chromatography using a phenyl resin to provide a saponin         composition.

Also provided is a method for the manufacture of a saponin composition comprising the steps of:

-   -   (i) treating a crude aqueous extract of Quillaja saponaria by         polyvinylpolypyrrolidone adsorption;     -   (ii) enzymatically modifying the treated extract with an         engineered glucosidase and an engineered rhamnosidase;     -   (iii) purifying the modified extract by reverse phase         chromatography using a polystyrene resin; and     -   (iv) purifying the modified extract by reverse phase         chromatography using a phenyl resin to provide a saponin         composition.

Typically, the crude aqueous extract is a bark extract. Suitably the QS-21 main peak content in an aqueous solution of crude aqueous extract of Quillaja saponaria is at least 1 g/L, such as at least 2 g/L, especially at least 2.5 g/L and in particular at least 2.8 g/L (e.g. as determined by UV absorbance relative to a control sample of known concentration).

The step of purifying the extract by polyvinylpolypyrrolidone adsorption involves treatment of the extract with polyvinylpolypyrrolidone adsorbant e.g. resin. Typically, the extract is agitated with the polyvinylpolypyrrolidone resin. The extract may subsequently be separated from the polyvinylpolypyrrolidone resin with adsorbed impurities by filtration. This step of the process generally removes polyphenolic impurities such as tannins.

The step of purifying the extract by reverse phase chromatography using a polystyrene resin typically uses acetonitrile and water as solvent, usually acidified with a suitable acid such as acetic acid. An example of a suitable resin is Amberchrom XT20. Chromatography may be undertaken using isocratic conditions, though is typically operated under a solvent gradient (continuous, such as linear, or stepped), such as those provided in the Examples. This step of the process generally removes non-saponin material and enriches the desired saponins. Each polystyrene chromatography run is typically at a scale of between 25-200 g of QS-21, such as between 50-150 g and in particular between 70-110 g (amounts being based on QS-21 main peak content in the material by UV).

Purifying the extract by reverse phase chromatography using a phenyl resin typically uses acetonitrile and water as solvent, usually acidified with a suitable acid such as acetic acid. Chromatography may be undertaken using a solvent gradient (continuous, such as linear, or stepped), though is typically operated under isocratic conditions. This step of the process provides the final purification of the desired saponins. Selected fractions may be pooled to maximise yield of material matching the required criteria. Each phenyl chromatography run is typically at a scale of between 4-40 g of QS-21, such as between 10-30 g and in particular between 13-21 g (amounts being based on QS-21 main peak content in the material by UV).

The method may comprise the further step of removing solvent to provide a dried saponin extract. Consequently, the invention provides a method for the manufacture of a saponin composition comprising the steps of:

-   -   (i) treating a crude aqueous extract of Quillaja saponaria by         polyvinylpolypyrrolidone adsorption;     -   (ii) enzymatically modifying the treated extract with a         glucosidase and/or a rhamnosidase;     -   (iii) purifying the modified extract by reverse phase         chromatography using a polystyrene resin; and     -   (iv) purifying the modified extract by reverse phase         chromatography using a phenyl resin; and     -   (v) removing solvent to provide a dried saponin composition.

The invention also provides a method for the manufacture of a saponin composition comprising the steps of:

-   -   (i) treating a crude aqueous extract of Quillaja saponaria by         polyvinylpolypyrrolidone adsorption;     -   (ii) enzymatically modifying the treated extract with an         engineered glucosidase polypeptide;     -   (iii) purifying the modified extract by reverse phase         chromatography using a polystyrene resin; and     -   (iv) purifying the modified extract by reverse phase         chromatography using a phenyl resin; and     -   (v) removing solvent to provide a dried saponin composition.

Further provided is a method for the manufacture of a saponin composition comprising the steps of:

-   -   (i) treating a crude aqueous extract of Quillaja saponaria by         polyvinylpolypyrrolidone adsorption;     -   (ii) enzymatically modifying the treated extract with an         engineered rhamnosidase polypeptide;     -   (iii) purifying the modified extract by reverse phase         chromatography using a polystyrene resin; and     -   (iv) purifying the modified extract by reverse phase         chromatography using a phenyl resin; and     -   (v) removing solvent to provide a dried saponin composition.

Additionally provided is a method for the manufacture of a saponin composition comprising the steps of:

-   -   (i) treating a crude aqueous extract of Quillaja saponaria by         polyvinylpolypyrrolidone adsorption;     -   (ii) enzymatically modifying the treated extract with an         engineered glucosidase polypeptide and an engineered         rhamnosidase polypeptide;     -   (iii) purifying the modified extract by reverse phase         chromatography using a polystyrene resin; and     -   (iv) purifying the modified extract by reverse phase         chromatography using a phenyl resin; and     -   (v) removing solvent to provide a dried saponin composition.

In order to improve drying efficiency, it may be desirable to undertake further steps of concentrating the extract, such as by capture and release using an appropriate technique, for example reverse phase chromatography (e.g. using a C8 resin), and/or exchanging the solvent in advance of the drying step.

Also provided is a method for the manufacture of a saponin composition comprising the steps:

-   -   (i) treating a crude aqueous extract of Quillaja saponaria by         polyvinylpolypyrrolidone adsorption;     -   (ii) enzymatically modifying the treated extract with a         glucosidase and/or a rhamnosidase;     -   (iii) purifying the modified extract by reverse phase         chromatography using a polystyrene resin; and     -   (iv) purifying the modified extract by reverse phase         chromatography using a phenyl resin;     -   (v) optionally concentrating the modified extract;     -   (vi) optionally exchanging the solvent; and     -   (vii) removing the remaining solvent to provide a dried saponin         composition;         wherein steps (v) and (vi) may be optionally be in reverse order         or undertaken concurrently, though are typically in the order         shown.

Further provided is a method for the manufacture of a saponin composition comprising the steps:

-   -   (i) treating a crude aqueous extract of Quillaja saponaria by         polyvinylpolypyrrolidone adsorption;     -   (ii) enzymatically modifying the treated extract with an         engineered glucosidase polypeptide;     -   (iii) purifying the modified extract by reverse phase         chromatography using a polystyrene resin; and     -   (iv) purifying the modified extract by reverse phase         chromatography using a phenyl resin;     -   (v) optionally concentrating the modified extract;     -   (vi) optionally exchanging the solvent; and     -   (vii) removing the remaining solvent to provide a dried saponin         composition;         wherein steps (v) and (vi) may be optionally be in reverse order         or undertaken concurrently, though are typically in the order         shown.

Additionally provided is a method for the manufacture of a saponin composition comprising the steps:

-   -   (i) treating a crude aqueous extract of Quillaja saponaria by         polyvinylpolypyrrolidone adsorption;     -   (ii) enzymatically modifying the treated extract with an         engineered rhamnosidase polypeptide;     -   (iii) purifying the modified extract by reverse phase         chromatography using a polystyrene resin; and     -   (iv) purifying the modified extract by reverse phase         chromatography using a phenyl resin;     -   (v) optionally concentrating the modified extract;     -   (vi) optionally exchanging the solvent; and     -   (vii) removing the remaining solvent to provide a dried saponin         composition;         wherein steps (v) and (vi) may be optionally be in reverse order         or undertaken concurrently, though are typically in the order         shown.

The invention provides a method for the manufacture of a saponin composition comprising the steps:

-   -   (i) treating a crude aqueous extract of Quillaja saponaria by         polyvinylpolypyrrolidone adsorption;     -   (ii) enzymatically modifying the treated extract with an         engineered glucosidase polypeptide and an engineered         rhamnosidase polypeptide;     -   (iii) purifying the modified extract by reverse phase         chromatography using a polystyrene resin; and     -   (iv) purifying the modified extract by reverse phase         chromatography using a phenyl resin;     -   (v) optionally concentrating the modified extract;     -   (vi) optionally exchanging the solvent; and     -   (vii) removing the remaining solvent to provide a dried saponin         composition;         wherein steps (v) and (vi) may be optionally be in reverse order         or undertaken concurrently, though are typically in the order         shown.

Also provided is a method for the manufacture of a saponin composition comprising the steps:

-   -   (i) treating a crude aqueous extract of Quillaja saponaria by         polyvinylpolypyrrolidone adsorption;     -   (ii) enzymatically modifying the treated extract with a         glucosidase and/or a rhamnosidase;     -   (iii) purifying the modified extract by diafiltration,         ultrafiltration or dialysis;     -   (iv) purifying the modified extract by reverse phase         chromatography using a polystyrene resin;     -   (v) purifying the modified extract by reverse phase         chromatography using a phenyl resin;     -   (vi) concentrating the modified extract by reverse phase         chromatography using a C8 resin;     -   (vii) exchanging the solvent; and     -   (viii) removing the remaining solvent to provide a dried saponin         composition.

Further provided is a method for the manufacture of a saponin composition comprising the steps:

-   -   (i) treating a crude aqueous extract of Quillaja saponaria by         polyvinylpolypyrrolidone adsorption;     -   (ii) enzymatically modifying the treated extract with an         engineered glucosidase polypeptide;     -   (iii) purifying the modified extract by diafiltration,         ultrafiltration or dialysis;     -   (iv) purifying the modified extract by reverse phase         chromatography using a polystyrene resin;     -   (v) purifying the modified extract by reverse phase         chromatography using a phenyl resin;     -   (vi) concentrating the modified extract by reverse phase         chromatography using a C8 resin;     -   (vii) exchanging the solvent; and     -   (viii) removing the remaining solvent to provide a dried saponin         composition.

Additionally provided is a method for the manufacture of a saponin composition comprising the steps:

-   -   (i) treating a crude aqueous extract of Quillaja saponaria by         polyvinylpolypyrrolidone adsorption;     -   (ii) enzymatically modifying the treated extract with an         engineered rhamnosidase polypeptide;     -   (iii) purifying the modified extract by diafiltration,         ultrafiltration or dialysis;     -   (iv) purifying the modified extract by reverse phase         chromatography using a polystyrene resin;     -   (v) purifying the modified extract by reverse phase         chromatography using a phenyl resin;     -   (vi) concentrating the modified extract by reverse phase         chromatography using a C8 resin;     -   (vii) exchanging the solvent; and     -   (viii) removing the remaining solvent to provide a dried saponin         composition.

The invention provides a method for the manufacture of a saponin composition comprising the steps:

-   -   (i) treating a crude aqueous extract of Quillaja saponaria by         polyvinylpolypyrrolidone adsorption;     -   (ii) enzymatically modifying the treated extract with an         engineered glucosidase polypeptide and an engineered         rhamnosidase polypeptide;     -   (iii) purifying the modified extract by diafiltration,         ultrafiltration or dialysis;     -   (iv) purifying the modified extract by reverse phase         chromatography using a polystyrene resin;     -   (v) purifying the modified extract by reverse phase         chromatography using a phenyl resin;     -   (vi) concentrating the modified extract by reverse phase         chromatography using a C8 resin;     -   (vii) exchanging the solvent; and     -   (viii) removing the remaining solvent to provide a dried saponin         composition.

The step of purifying the extract by diafiltration, ultrafiltration or dialysis, is suitably purification by diafiltration. typically using tangential flow. An appropriate example of a membrane is a 30 kDa cut-off. This step of the process generally removes salts, sugars and other low molecular weight materials.

Concentration of the extract may be performed using any suitable technique. For example, concentration may be performed using a capture and release methodology, such as reverse phase chromatography, in particular using a C8 resin. The reverse phase chromatography typically uses acetonitrile and water as solvent, usually acidified with a suitable acid such as acetic acid. Chromatography is typically operated under a solvent gradient, with the saponin extract captured in low organic solvent and eluted in high organic solvent, in particular, a stepped solvent gradient.

Exchanging the solvent may be performed using any suitable technique, in particular diafiltration, ultrafiltration or dialysis, especially diafiltration. Solvent exchange may be useful, for example, in reducing the acetonitrile content such as described in WO2014016374. A suitable membrane may be selected to allow solvent exchange while retaining the saponin extract, such as a 1 kDa membrane.

Drying, by removing the solvent, may be undertaken by any suitable means, in particular by lyophilisation. During drying, degradation of the saponin extract can occur, leading to the formation of Iyo impurity. Consequently, it is desirable to dry under conditions which limit formation of Iyo impurity, such as by limiting the drying temperature and/or drying time. Suitably removal of solvent is undertaken by a single lyophilisation process. The extent of drying required will depend on the nature of the solvent, for example non-pharmaceutically acceptable solvents will desirably be removed to a high degree, whereas some pharmaceutically acceptable solvents (such as water) may be removed to a lesser degree.

Suitably the methods of the present invention are undertaken at a scale of between 25-1000 g of QS-21, such as between 50-500 g and in particular between 100-500 g (amounts being based on QS-21 main peak content in the material by UV).

Provided is a product saponin prepared according to the present invention. There is provided the use of a product saponin prepared according to the present invention in the manufacture of a medicament. Additionally, provided is a product saponin prepared according to the present invention for use as a medicament, in particular as an adjuvant. Also provided is an adjuvant composition comprising a product saponin prepared according to the present invention.

There is provided a crude extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a glucosidase. There is also provided a crude extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a rhamnosidase. Also provided is a crude extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a glucosidase and a rhamnosidase.

There is provided a crude bark extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a glucosidase. There is also provided a crude bark extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a rhamnosidase. Also provided is a crude saponin extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a glucosidase and a rhamnosidase.

There is provided a PVPP treated extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a glucosidase. There is also provided a PVPP treated extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a rhamnosidase. Also provided is a PVPP treated extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a glucosidase and a rhamnosidase.

There is provided a PVPP treated bark extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a glucosidase. There is also provided a PVPP treated bark extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a rhamnosidase. Also provided is a PVPP treated saponin extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a glucosidase and a rhamnosidase.

Also provided is a saponin composition containing at least 93% QS-21 main peak and <0.25% 2018 component by UV absorbance at 214 nm. Suitably wherein the monoisotope of the most abundant species is 1987.9 m/z. Desirably, the saponin composition contains at least 98% QS-21 group by UV absorbance at 214 nm. Desirably, the extract contains 1% or less of Iyo impurity by UV absorbance at 214 nm. Desirably, the extract contains 1% or less of largest peak outside the QS-21 group by UV absorbance at 214 nm.

Also provided is a saponin composition containing at least 98% QS-21 group, at least 93% QS-21 main peak, <0.25% 2018 component, 1% or less of largest peak outside the QS-21 group by UV absorbance at 214 nm and wherein the monoisotope of the most abundant species is 1987.9 m/z. Suitably the saponin composition contains <0.23% 2018 component, especially <0.21% 2018 component, in particular <0.21% 2018 component, such as 0.2% or less 2018 component.

The saponin compositions desirably comprise at least 40%, such as at least 50%, suitably at least 60%, especially at least 70% and desirably at least 80%, for example at least 90% (as determined by UV absorbance at 214 nm and by relative ion abundance) QS-21 1988 A component, QS-21 1856 A component and/or QS-21 2002 A component. In certain embodiments, the saponin composition comprises at least 40%, such as at least 50%, in particular at least 60%, especially at least 65%, such as at least 70%, QS-21 1988 A component as determined by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments the saponin composition contain 90% or less, such as 85% or less, or 80% or less, QS-21 1988 A component as determined by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments, the saponin composition contain from 40% to 90% QS-21 1988 A component, such as 50% to 85% QS-21 1988 A component, especially 70% to 80% QS-21 1988 A component as determined by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments, the saponin compositions contain 30% or less, such as 25% or less, QS-21 1856 A as determined by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments the saponin composition contain at least 5%, such as at least 10% QS-21 1856 A by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments, the saponin compositions contain from 5% to 30% QS-21 1856 A, such as 10% to 25% QS-21 1856 A as determined by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments, the saponin composition contains 40% or less, such as 30% or less, in particular 20% or less, especially 10% or less QS-21 2002 A component by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments, the saponin composition contain at least 0.5%, such as at least 1%, QS-21 2002 A component by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments, the saponin composition contain from 0.5% to 40% QS-21 2002 A component, such as 1% to 10% QS-21 2002 A component as determined by UV absorbance at 214 nm and by relative ion abundance.

By the term ‘Iyo impurity’ is meant the triterpenoid glycosides identified as ‘Lyophilization Peak’ in FIG. 6 . Suitably the Iyo impurity in the UPLC-UV/MS methods described herein has a retention time of approximately 4.7 min and the primary component of the peak having a monoisotopic molecular weight of 1855.9. The terms 2018 component, QS-21 main peak, QS-21 group may be understood such as by reference to the examples herein.

The saponin compositions of the present invention (i.e. a composition comprising a product saponin prepared according to the present invention) may be combined with further adjuvants, such as a TLR4 agonist, in particular lipopolysaccharide TLR4 agonists, such as lipid A derivatives, especially a monophosphoryl lipid A e.g. 3-de-O-acylated monophosphoryl lipid A (3D-MPL). 3D-MPL is sold under the name ‘MPL’ by GlaxoSmithKline Biologicals N.A. and is referred throughout the document as 3D-MPL. See, for example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094. 3D-MPL can be produced according to the methods described in GB 2 220 211 A. Chemically it is a mixture of 3-deacylated monophosphoryl lipid A with 4, 5 or 6 acylated chains.

Other TLR4 agonists which may be of use in the present invention include Glucopyranosyl Lipid Adjuvant (GLA) such as described in WO2008/153541 or WO2009/143457 or the literature articles Coler R N et al. (2011) Development and Characterization of Synthetic Glucopyranosyl Lipid Adjuvant System as a Vaccine Adjuvant. PLoS ONE 6(1): e16333. doi:10.1371/journal.pone.0016333 and Arias M A et al. (2012) Glucopyranosyl Lipid Adjuvant (GLA), a Synthetic TLR4 Agonist, Promotes Potent Systemic and Mucosal Responses to Intranasal Immunization with HIVgp140. PLoS ONE 7(7): e41144. doi:10.1371/journal.pone.0041144. WO2008/153541 or WO2009/143457 are incorporated herein by reference for the purpose of defining TLR4 agonists which may be of use in the present invention.

A particular alkyl glucosaminide phosphate (AGP) of interest is set forth as follows:

TLR4 agonists of interest include:

3-deacyl monophosphoryl hexa-acyl lipid A.

Another TLR4 agonist of interest is:

3-deacyl monophosphoryl lipid A.

A TLR4 agonist of interest is dLOS (as described in Han, 2014):

A typical adult human dose of adjuvant will comprise a saponin composition, such as a Q-21 composition, at amounts between 1 and 100 ug per human dose. The saponin extract may be used at a level of about 50 ug. Examples of suitable ranges are 40-60 ug, suitably 45-ug or 49-51 ug, such as 50 ug. In a further embodiment, the human dose comprises saponin composition, such as a Q-21 composition, at a level of about 25 ug. Examples of lower ranges include 20-30 ug, suitably 22-28 ug or 24-26 ug, such as 25 ug. Human doses intended for children may be reduced compared to those intended for an adult (e.g. reduction by 50%).

The TLR4 agonists, such as a lipopolysaccharide, such as 3D-MPL, can be used at amounts between 1 and 100 ug per human dose. 3D-MPL may be used at a level of about 50 ug. Examples of suitable ranges are 40-60 ug, suitably 45-55 ug or 49-51 ug, such as 50 ug. In a further embodiment, the human dose comprises 3D-MPL at a level of about 25 ug. Examples of lower ranges include 20-30 ug, suitably 22-28 ug or 24-26 ug, such as 25 ug. Human doses intended for children may be reduced compared to those intended for an adult (e.g. reduction by 50%).

When both a TLR4 agonist and a saponin composition, such as a Q-21 composition, are present in the adjuvant, then the weight ratio of TLR4 agonist to saponin is suitably between 1:5 to 5:1, suitably 1:1. For example, where 3D-MPL is present at an amount of 50 ug or 25 ug, then suitably QS-21 may also be present at an amount of 50 ug or 25 ug per human dose.

Adjuvants may also comprise a suitable carrier, such as an emulsion (e.g. an oil in water emulsion, such as a squalene containing oil in water emulsion) or liposomes.

The present invention provides an adjuvant composition comprising a saponin composition according to the present invention. Suitably the adjuvant composition further comprises a TLR4 agonist.

Liposomes

The term ‘liposome’ is well known in the art and defines a general category of vesicles which comprise one or more lipid bilayers surrounding an aqueous space. Liposomes thus consist of one or more lipid and/or phospholipid bilayers and can contain other molecules, such as proteins or carbohydrates, in their structure. Because both lipid and aqueous phases are present, liposomes can encapsulate or entrap water-soluble material, lipid-soluble material, and/or amphiphilic compounds.

Liposome size may vary from 30 nm to several um depending on the phospholipid composition and the method used for their preparation.

The liposomes of use in the present invention suitably contain DOPC, or, consist essentially of DOPC and sterol (with saponin and optionally TLR4 agonist).

In the present invention, the liposome size will be in the range of 50 nm to 200 nm, especially 60 nm to 180 nm, such as 70-165 nm. Optimally, the liposomes should be stable and have a diameter of ˜100 nm to allow convenient sterilization by filtration.

Structural integrity of the liposomes may be assessed by methods such as dynamic light scattering (DLS) measuring the size (Z-average diameter, Zav) and polydispersity of the liposomes, or, by electron microscopy for analysis of the structure of the liposomes. In one embodiment the average particle size is between 95 and 120 nm, and/or, the polydispersity (Pdl) index is not more than 0.3 (such as not more than 0.2).

Further Excipients

In a further embodiment, a buffer is added to an adjuvant composition. The pH of a liquid preparation is adjusted in view of the components of the composition and necessary suitability for administration to the subject. Suitably, the pH of a liquid mixture is at least 4, at least 5, at least 5.5, at least 5.8, at least 6. The pH of the liquid mixture may be less than 9, less than 8, less than 7.5 or less than 7. In other embodiments, pH of the liquid mixture is between 4 and 9, between 5 and 8, such as between 5.5 and 8. Consequently, the pH will suitably be between 6-9, such as 6.5-8.5. In a particularly preferred embodiment the pH is between 5.8 and 6.4. An appropriate buffer may be selected from acetate, citrate, histidine, maleate, phosphate, succinate, tartrate and TRIS. In one embodiment, the buffer is a phosphate buffer such as Na/Na₂PO₄, Na/K₂PO₄ or K/K₂PO₄.

The buffer can be present in the liquid mixture in an amount of at least 6 mM, at least 10 mM or at least 40 mM. The buffer can be present in the liquid mixture in an amount of less than 100 mM, less than 60 mM or less than 40 mM.

It is well known that for parenteral administration solutions should have a pharmaceutically acceptable osmolality to avoid cell distortion or lysis. A pharmaceutically acceptable osmolality will generally mean that solutions will have an osmolality which is approximately isotonic or mildly hypertonic. Suitably the compositions (when reconstituted, if presented in dried form) will have an osmolality in the range of 250 to 750 mOsm/kg, for example, the osmolality may be in the range of 250 to 550 mOsm/kg, such as in the range of 280 to 500 mOsm/kg. In a particularly preferred embodiment the osmolality may be in the range of 280 to 310 mOsm/kg. Osmolality may be measured according to techniques known in the art, such as by the use of a commercially available osmometer, for example the Advanced® Model 2020 available from Advanced Instruments Inc. (USA).

An “isotonicity agent” is a compound that is physiologically tolerated and imparts a suitable tonicity to a formulation to prevent the net flow of water across cell membranes that are in contact with the formulation. In some embodiments, the isotonicity agent used for the composition is a salt (or mixtures of salts), conveniently the salt is sodium chloride, suitably at a concentration of approximately 150 nM. In other embodiments, however, the composition comprises a non-ionic isotonicity agent and the concentration of sodium chloride in the composition is less than 100 mM, such as less than 80 mM, e.g. less than 50 mM, such as less mM, less than 30 mM and especially less than 20 mM. The ionic strength in the composition may be less than 100 mM, such as less than 80 mM, e.g. less than 50 mM, such as less 40 mM or less than 30 mM.

In a particular embodiment, the non-ionic isotonicity agent is a polyol, such as sucrose and/or sorbitol. The concentration of sorbitol may e.g. between about 3% and about 15% (w/v), such as between about 4% and about 10% (w/v). Adjuvants comprising an immunologically active saponin fraction and a TLR4 agonist wherein the isotonicity agent is salt or a polyol have been described in WO2012/080369.

Suitably, a human dose volume of between 0.05 ml and 1 ml, such as between 0.1 and ml, in particular a dose volume of about 0.5 ml, or 0.7 ml. The volumes of the compositions used may depend on the delivery route and location, with smaller doses being given by the intradermal route. A unit dose container may contain an overage to allow for proper manipulation of materials during administration of the unit dose.

The ratio of saponin:DOPC will typically be in the order of 1:50 to 1:10 (w/w), suitably between 1:25 to 1:15 (w/w), and preferably 1:22 to 1:18 (w/w), such as 1:20 (w/w).

Suitably the saponin is presented in a less reactogenic composition where it is quenched with an exogenous sterol, such as cholesterol. Cholesterol is disclosed in the Merck Index, 13th Edn., page 381, as a naturally occurring sterol found in animal fat. Cholesterol has the formula (C₂₇H₄₆O) and is also known as (3β)-cholest-5-en-3-ol.

The ratio of saponin:sterol will typically be in the order of 1:100 to 1:1 (w/w), suitably between 1:10 to 1:1 (w/w), and preferably 1:5 to 1:1 (w/w). Suitably excess sterol is present, the ratio of saponin:sterol being at least 1:2 (w/w). In one embodiment, the ratio of saponin:sterol is 1:5 (w/w). In one embodiment, the sterol is cholesterol.

The amount of liposome (weight of lipid and sterol) will typically be in the range of 0.1 mg to 10 mg per human dose of a composition, in particular 0.5 mg to 2 mg per human dose of a composition.

In a particularly suitable embodiment, liposomes used in the invention comprise DOPC and a sterol, in particular cholesterol. Thus, in a particular embodiment, a composition used in the invention comprises saponin extract in the form of a liposome, wherein said liposome comprises DOPC and a sterol, in particular cholesterol.

A particular adjuvant of interest features liposomes comprising DOPC and cholesterol, with TLR4 agonist and a saponin prepared according to the present invention, especially 3D-MPL and a saponin prepared according to the present invention.

Another adjuvant of interest features liposomes comprising DOTAP and DMPC, with TLR4 agonist and a saponin prepared according to the present invention, especially dLOS and a saponin prepared according to the present invention.

Antigens

The adjuvants prepared according to the present invention may be utilised in conjunction with an immunogen or antigen. In some embodiments a polynucleotide encoding the immunogen or antigen is provided.

The adjuvant may be administered to a subject separately from an immunogen or antigen, or the adjuvant may be combined, either during manufacturing or extemporaneously, with an immunogen or antigen to provide an immunogenic composition for combined administration.

As used herein, a subject is a mammalian animal, such as a rodent, non-human primate, or human.

Consequently, there is provided a method for the preparation of an immunogenic composition comprising an immunogen or antigen, or a polynucleotide encoding the immunogen or antigen, said method comprising the steps of:

-   -   (i) preparing an adjuvant composition comprising a saponin         prepared according to the present invention;     -   (ii) mixing the adjuvant with an immunogen or antigen, or a         polynucleotide encoding the immunogen or antigen.

There is also provided the use of an adjuvant comprising a saponin prepared according to the present invention in the manufacture of a medicament. Suitably the medicament comprises an immunogen or antigen, or a polynucleotide encoding the immunogen or antigen. Further provided is an adjuvant comprising a saponin prepared according to the present invention for use as a medicament. Suitably the medicament comprises an immunogen or antigen, or a polynucleotide encoding the immunogen or antigen.

By the term immunogen is meant a polypeptide which is capable of eliciting an immune response. Suitably the immunogen is an antigen which comprises at least one B or T cell epitope. The elicited immune response may be an antigen specific B cell response, which produces neutralizing antibodies. The elicited immune response may be an antigen specific T cell response, which may be a systemic and/or a local response. The antigen specific T cell response may comprise a CD4+ T cell response, such as a response involving CD4+ T cells expressing a plurality of cytokines, e.g. IFNgamma, TNFalpha and/or IL2. Alternatively, or additionally, the antigen specific T cell response comprises a CD8+ T cell response, such as a response involving CD8+ T cells expressing a plurality of cytokines, e.g., IFNgamma, TNFalpha and/or IL2.

The antigen may be derived (such as obtained from) from a human or non-human pathogen including, e.g., bacteria, fungi, parasitic microorganisms or multicellular parasites which infect human and non-human vertebrates, or from a cancer cell or tumor cell.

In one embodiment the antigen is a recombinant protein, such as a recombinant prokaryotic protein.

A plurality of antigens may be provided. For example, a plurality of antigens may be provided to strengthen the elicited immune response (e.g. to ensure strong protection), a plurality of antigens may be provided to broaden the immune response (e.g. to ensure protection against a range of pathogen strains or in a large proportion of a subject population) or a plurality of antigens may be provided to currently elicit immune responses in respect of a number of disorders (thereby simplifying administration protocols). Where a plurality of antigens are provided, these may be as distinct proteins or may be in the form of one or more fusion proteins.

Antigen may be provided in an amount of 0.1 to 100 ug per human dose. The present invention may be applied for use in the treatment or prophylaxis of a disease or disorder associated with one or more antigens described above. In one embodiment the disease or disorder is selected from malaria, tuberculosis, COPD, HIV and herpes.

The adjuvant may be administered separately from an immunogen or antigen, or may be combined, either during manufacturing or extemporaneously), with an immunogen or antigen to provide an immunogenic composition for combined administration.

Sterilisation

For parenteral administration in particular, compositions should be sterile. Sterilisation can be performed by various methods although is conveniently undertaken by filtration through a sterile grade filter. Sterilisation may be performed a number of times during preparation of an adjuvant or immunogenic composition, but is typically performed at least at the end of manufacture.

By “sterile grade filter” it is meant a filter that produces a sterile effluent after being challenged by microorganisms at a challenge level of greater than or equal to 1×10⁷/cm² of effective filtration area. Sterile grade filters are well known to the person skilled in the art of the invention for the purpose of the present invention, sterile grade filters have a pore size between and 0.25 um, suitably 0.18-0.22 um, such as 0.2 or 0.22 um.

The membranes of the sterile grade filter can be made from any suitable material known to the skilled person, for example, but not limited to cellulose acetate, polyethersulfone (PES), polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE). In a particular embodiment of the invention one or more or all of the filter membranes of the present invention comprise polyethersulfone (PES), in particular hydrophilic polyethersulfone. In a particular embodiment of the invention, the filters used in the processes described herein are a double layer filter, in particular a sterile filter with built-in prefilter having larger pore size than the pore size of the end filter. In one embodiment the sterilizing filter is a double layer filter wherein the pre-filter membrane layer has a pore size between 0.3 and 0.5 nm, such as 0.35 or 0.45 nm. According to further embodiments, filters comprise asymmetric filter membrane(s), such as asymmetric hydrophilic PES filter membrane(s). Alternatively, the sterilizing filter layer may be made of PVDF, e.g. in combination with an asymmetric hydrophilic PES pre-filter membrane layer. In light of the intended medical uses, materials should be of pharmaceutical grade (such as parenteral grade).

Clauses of the Invention

The invention is illustrated by the following clauses:

-   -   Clause 1. A method for making a product saponin, said method         comprising the step of enzymatically converting a starting         saponin to the product saponin.     -   Clause 2. A method for increasing the amount of a product         saponin in a composition, said method comprising the step of         enzymatically converting a starting saponin to the product         saponin.     -   Clause 3. A method for reducing the amount of a starting saponin         in a composition, said method comprising the step of         enzymatically converting the starting saponin to a product         saponin.     -   Clause 4. The method according to any one of clauses 1 to 3,         wherein the starting saponin is a naturally occurring saponin.     -   Clause 5. The method according to any one of clauses 1 to 3,         wherein the starting saponin is an artificial saponin.     -   Clause 6. The method according to any one of clauses 1 to 5,         wherein the starting saponin is a steroid glycoside.     -   Clause 7. The method according to any one of clauses 1 to 6,         wherein the starting saponin is a terpenoid glycoside.     -   Clause 8. The method according to clause 7, wherein the starting         saponin is a triterpenoid glycoside.     -   Clause 9. The method according to clause 8, wherein the starting         saponin is a quillaic acid glycoside.     -   Clause 10. The method according to any one of clauses 1 to 4 or         6 to 9, wherein the starting saponin is obtainable from plants         of the genera Gypsophilia, Saponaria or Quillaja.     -   Clause 11. The method according to clause 10, wherein the         starting saponin is obtainable from Quillaja species.     -   Clause 12. The method according to clause 11, wherein the         starting saponin is obtainable from Quillaja brasiliensis.     -   Clause 13. The method according to clause 12, wherein the         starting saponin is obtained from Quillaja brasiliensis.     -   Clause 14. The method according to clause 11, wherein the         starting saponin is obtainable from Quillaja saponaria.     -   Clause 15. The method according to clause 14, wherein the         starting saponin is obtained from Quillaja saponaria.     -   Clause 16. The method according to either clause 14 or 15,         wherein the starting saponin is a QS-18 family component.     -   Clause 17. The method according to clause 16, wherein the         starting saponin is a QS-18 2150 A component.     -   Clause 18. The method according to clause 17, wherein the         starting saponin is QS-18 2150 A V1.     -   Clause 19. The method according to clause 17, wherein the         starting saponin is QS-18 2150 A V2.     -   Clause 20. The method according to clause 16, wherein the         starting saponin is QS-18 2018 A component.     -   Clause 21. The method according to clause 16, wherein the         starting saponin is a QS-18 2164 A component.     -   Clause 22. The method according to clause 21, wherein the         starting saponin is QS-18 2164 A V1.     -   Clause 23. The method according to clause 21, wherein the         starting saponin is QS-18 2164 A V2.     -   Clause 24. The method according to clause 16, wherein the         starting saponin is a QS-18 2150 B component.     -   Clause 26. The method according to clause 24, wherein the         starting saponin is QS-18 2150 B V1.     -   Clause 26. The method according to clause 24, wherein the         starting saponin is QS-18 2150 B V2.     -   Clause 27. The method according to clause 16, wherein the         starting saponin is QS-18 2018 B component.     -   Clause 28. The method according to clause 16, wherein the         starting saponin is a QS-18 2164 B component.     -   Clause 29. The method according to clause 28, wherein the         starting saponin is QS-18 2164 B V1.     -   Clause 30. The method according to clause 28, wherein the         starting saponin is QS-18 2164 B V2.     -   Clause 31. The method according to clause 15, wherein the         starting saponin is a desglucosyl-QS-17 family component.     -   Clause 32. The method according to clause 31, wherein the         starting saponin is a desglucosyl-QS-17 2134 A component.     -   Clause 33. The method according to clause 32, wherein the         starting saponin is desglucosyl-QS-17 2134 A V1.     -   Clause 34. The method according to clause 32, wherein the         starting saponin is desglucosyl-QS-17 2134 A V2.     -   Clause 35. The method according to clause 31, wherein the         starting saponin is desglucosyl-QS-17 2002 A component.     -   Clause 36. The method according to clause 31, wherein the         starting saponin is a desglucosyl-QS-17 2148 A component.     -   Clause 37. The method according to clause 36, wherein the         starting saponin is desglucosyl-QS-17 2148 A V1.     -   Clause 38. The method according to clause 36, wherein the         starting saponin is desglucosyl-QS-17 2148 A V2.     -   Clause 39. The method according to clause 31, wherein the         starting saponin is a desglucosyl-QS-17 2134 B component.     -   Clause 40. The method according to clause 39, wherein the         starting saponin is desglucosyl-QS-17 2134 B V1.     -   Clause 41. The method according to clause 39, wherein the         starting saponin is desglucosyl-QS-17 2134 B V2.     -   Clause 42. The method according to clause 31, wherein the         starting saponin is desglucosyl-QS-17 2002 B component.     -   Clause 43. The method according to clause 31, wherein the         starting saponin is a desglucosyl-QS-17 2148 B component.     -   Clause 44. The method according to clause 43, wherein the         starting saponin is desglucosyl-QS-17 2148 B V1.     -   Clause 45. The method according to clause 43, wherein the         starting saponin is desglucosyl-QS-17 2148 B V2.     -   Clause 46. The method according to clause 15, wherein the         starting saponin is a QS-17 family component.     -   Clause 47. The method according to clause 46, wherein the         starting saponin is a QS-17 2296 A component.     -   Clause 48. The method according to clause 47, wherein the         starting saponin is QS-17 2296 A V1.     -   Clause 49. The method according to clause 48, wherein the         starting saponin is QS-17 2296 A V2.     -   Clause 50. The method according to clause 46, wherein the         starting saponin is QS-17 2164 A component.     -   Clause 51. The method according to clause 46, wherein the         starting saponin is a QS-17 2310 A component.     -   Clause 52. The method according to clause 51, wherein the         starting saponin is QS-17 2310 A V1.     -   Clause 53. The method according to clause 51, wherein the         starting saponin is QS-17 2310 A V2.     -   Clause 54. The method according to clause 46, wherein the         starting saponin is a QS-17 2296 B component.     -   Clause 55. The method according to clause 54, wherein the         starting saponin is QS-17 2296 B V1.     -   Clause 56. The method according to clause 54, wherein the         starting saponin is QS-17 2296 B V2.     -   Clause 57. The method according to clause 46, wherein the         starting saponin is QS-17 2164 B component.     -   Clause 58. The method according to clause 46, wherein the         starting saponin is a QS-17 2310 B component.     -   Clause 59. The method according to clause 58, wherein the         starting saponin is QS-17 2310 B V1     -   Clause 60. The method according to clause 58, wherein the         starting saponin is QS-17 2310 B V2.     -   Clause 61. The method according to clause 15, wherein the         starting saponin is a desarabinofuranosyl-QS-18 family         component.     -   Clause 62. The method according to clause 61, wherein the         starting saponin is a desarabinofuranosyl-QS-18 2018 A         component.     -   Clause 63. The method according to clause 62, wherein the         starting saponin is desarabinofuranosyl-QS-18 2018 A V1.     -   Clause 64. The method according to clause 62, wherein the         starting saponin is desarabinofuranosyl-QS-18 2018 A V2.     -   Clause 65. The method according to clause 61, wherein the         starting saponin is desarabinofuranosyl-QS-18 1886 A component.     -   Clause 66. The method according to clause 61, wherein the         starting saponin is a desarabinofuranosyl-QS-18 2032 A         component.     -   Clause 67. The method according to clause 65, wherein the         starting saponin is desarabinofuranosyl-QS-18 2032 A V1.     -   Clause 68. The method according to clause 65, wherein the         starting saponin is desarabinofuranosyl-QS-18 2032 A V2.     -   Clause 69. The method according to clause 61, wherein the         starting saponin is a desarabinofuranosyl-QS-18 2018 B         component.     -   Clause 70. The method according to clause 69, wherein the         starting saponin is desarabinofuranosyl-QS-18 2018 B V1.     -   Clause 71. The method according to clause 69, wherein the         starting saponin is desarabinofuranosyl-QS-18 2018 B V2.     -   Clause 72. The method according to clause 61, wherein the         starting saponin is desarabinofuranosyl-QS-18 1886 B component.     -   Clause 73. The method according to clause 61, wherein the         starting saponin is a desarabinofuranosyl-QS-18 2032 B         component.     -   Clause 74. The method according to clause 73, wherein the         starting saponin is desarabinofuranosyl-QS-18 2032 B V1.     -   Clause 75. The method according to clause 73, wherein the         starting saponin is desarabinofuranosyl-QS-18 2032 B V2.     -   Clause 76. The method according to clause 15, wherein the         starting saponin is an acetylated desglucosyl-QS-17 family         component.     -   Clause 77. The method according to clause 76, wherein the         starting saponin is an acetylated desglucosyl-QS-17 2176 A         component.     -   Clause 78. The method according to clause 77, wherein the         starting saponin is acetylated desglucosyl-QS-17 2176 A V1.     -   Clause 79. The method according to clause 77, wherein the         starting saponin is acetylated desglucosyl-QS-17 2176 A V2.     -   Clause 80. The method according to clause 76, wherein the         starting saponin is acetylated desglucosyl-QS-17 2044 A         component.     -   Clause 81. The method according to clause 76, wherein the         starting saponin is an acetylated desglucosyl-QS-17 2190 A         component.     -   Clause 82. The method according to clause 81, wherein the         starting saponin is acetylated desglucosyl-QS-17 2190 A V1.     -   Clause 83. The method according to clause 81, wherein the         starting saponin is acetylated desglucosyl-QS-17 2190 A V2.     -   Clause 84. The method according to any one of clauses 1 to 83,         wherein the product saponin is a naturally occurring saponin.     -   Clause 85. The method according to any one of clauses 1 to 83,         wherein the product saponin is an artificial saponin.     -   Clause 86. The method according to any one of clauses 1 to 85,         wherein the product saponin is a steroid glycoside.     -   Clause 87. The method according to any one of clauses 1 to 85,         wherein the product saponin is a terpenoid glycoside.     -   Clause 88. The method according to clause 87, wherein the         product saponin is a triterpenoid glycoside.     -   Clause 89. The method according to clause 88, wherein the         product saponin is a quillaic acid glycoside.     -   Clause 90. The method according to any one of clauses 1 to 84 or         86 to 89, wherein the product saponin is obtainable from plants         of the genera Gypsophilia, Saponaria or Quillaja.     -   Clause 91. The method according to clause 90, wherein the         product saponin is obtainable Quillaja species.     -   Clause 92. The method according to clause 91, wherein the         product saponin is obtainable from Quillaja brasiliensis.     -   Clause 93. The method according to clause 92, wherein the         product saponin is obtainable from Quillaja saponaria.     -   Clause 94. The method according to any one of clauses 16, 31 or         93, wherein the product saponin is a QS-21 family component.     -   Clause 95. The method according to any one of clauses 17, 32 or         94, wherein the product saponin is a QS-21 1988 A component.     -   Clause 96. The method according to any one of clauses 18, 33 or         95, wherein the product saponin is QS-21 1988 A V1.     -   Clause 97. The method according to any one of clauses 19, 34 or         95, wherein the product saponin is QS-21 1988 A V2.     -   Clause 98. The method according to any one of clauses 20, 35 or         94, wherein the product saponin is QS-21 1856 A component.     -   Clause 99. The method according to any one of clauses 21, 36 or         94, wherein the product saponin is a QS-21 2002 A component.     -   Clause 100. The method according to any one of clauses 22, 37 or         99, wherein the product saponin is QS-21 2002 A V1.     -   Clause 101. The method according to any one of clauses 23, 38 or         99, wherein the product saponin is QS-21 2002 A V2.     -   Clause 102. The method according to any one of clauses 24, 39 or         94, wherein the product saponin is a QS-21 1988 B component.     -   Clause 103. The method according to any one of clauses 25, 40 or         102, wherein the product saponin is QS-21 1988 B V1.     -   Clause 104. The method according to any one of clauses 26, 41 or         102, wherein the product saponin is QS-21 1988 B V2.     -   Clause 105. The method according to any one of clauses 27, 42 or         94, wherein the product saponin is QS-21 1856 B component.     -   Clause 106. The method according to any one of clauses 28, 43 or         94, wherein the product saponin is a QS-21 2002 B component.     -   Clause 107. The method according to any one of clauses 29, 44 or         106, wherein the product saponin is QS-21 2002 B V1.     -   Clause 108. The method according to any one of clauses 30, 45 or         106, wherein the product saponin is QS-21 2002 B V2.     -   Clause 109. The method according to either clause 46 or 93,         wherein the product saponin is a QS-18 family component.     -   Clause 110. The method according to either clause 47 or 109,         wherein the product saponin is a QS-18 2150 A component.     -   Clause 111. The method according to either clause 48 or 110,         wherein the product saponin is QS-18 2150 A V1.     -   Clause 112. The method according to either clause 49 or 110,         wherein the product saponin is QS-18 2150 A V2.     -   Clause 113. The method according to either clause 50 or 109,         wherein the product saponin is QS-18 2018 A component.     -   Clause 114. The method according to either clause 51 or 109,         wherein the product saponin is a QS-18 2164 A component.     -   Clause 115. The method according to either clause 52 or 114,         wherein the product saponin is QS-18 2164 A V1.     -   Clause 116. The method according to either clause 53 or 114,         wherein the product saponin is QS-18 2164 A V2.     -   Clause 117. The method according to either clause 54 or 109,         wherein the product saponin is a QS-18 2150 B component.     -   Clause 118. The method according to either clause 55 or 117,         wherein the product saponin is QS-18 2150 B V1.     -   Clause 119. The method according to either clause 56 or 117,         wherein the product saponin is QS-18 2150 B V2.     -   Clause 120. The method according to either clause 57 or 109,         wherein the product saponin is QS-18 2018 B component.     -   Clause 121. The method according to either clause 58 or 120,         wherein the product saponin is a QS-18 2164 B component.     -   Clause 122. The method according to either clause 59 or 121,         wherein the product saponin is QS-18 2164 B V1.     -   Clause 123. The method according to either clause 60 or 121,         wherein the product saponin is QS-18 2164 B V2.     -   Clause 124. The method according to either clause 46 or 93,         wherein the product saponin is a desglucosyl-QS-17 family         component.     -   Clause 125. The method according to either clause 47 or 124,         wherein the product saponin is a desglucosyl-QS-17 2134 A         component.     -   Clause 126. The method according to either clause 48 or 125,         wherein the product saponin is desglucosyl-QS-17 2134 A V1.     -   Clause 127. The method according to either clause 49 or 125,         wherein the product saponin is desglucosyl-QS-17 2134 A V2.     -   Clause 128. The method according to either clause 50 or 124,         wherein the product saponin is desglucosyl-QS-17 2002 A         component.     -   Clause 129. The method according to either clause 51 or 124,         wherein the product saponin is a desglucosyl-QS-17 2148 A         component.     -   Clause 130. The method according to either clause 52 or 129,         wherein the product saponin is desglucosyl-QS-17 2148 A V1.     -   Clause 131. The method according to either clause 53 or 129,         wherein the product saponin is desglucosyl-QS-17 2148 A V2.     -   Clause 132. The method according to either clause 54 or 124,         wherein the product saponin is a desglucosyl-QS-17 2134 B         component.     -   Clause 133. The method according to either clause 55 or 132,         wherein the product saponin is desglucosyl-QS-17 2134B V1.     -   Clause 134. The method according to either clause 56 or 132,         wherein the product saponin is desglucosyl-QS-17 2134 B V2.     -   Clause 135. The method according to either clause 57 or 124,         wherein the product saponin is desglucosyl-QS-17 2002 B         component.     -   Clause 136. The method according to either clause 58 or 124,         wherein the product saponin is a desglucosyl-QS-17 2148 B         component.     -   Clause 137. The method according to either clause 59 or 136,         wherein the product saponin is desglucosyl-QS-17 2148 B V1.     -   Clause 138. The method according to either clause 60 or 136,         wherein the product saponin is desglucosyl-QS-17 2148 B V2.     -   Clause 140. The method according to either clause 61 or 93,         wherein the product saponin is a desarabinofuranosyl-QS-21         family component.     -   Clause 140. The method according to either clause 62 or 139,         wherein the product saponin is a desarabinofuranosyl-QS-21 1856         A component.     -   Clause 141. The method according to either clause 63 or 140,         wherein the product saponin is desarabinofuranosyl-QS-21 1856 A         V1.     -   Clause 142. The method according to either clause 64 or 140,         wherein the product saponin is desarabinofuranosyl-QS-21 1856 A         V2.     -   Clause 143. The method according to either clause 65 or 139,         wherein the product saponin is desarabinofuranosyl-QS-21 1712 A         component.     -   Clause 144. The method according to either clause 66 or 139,         wherein the product saponin is a desarabinofuranosyl-QS-21 1870         A component.     -   Clause 145. The method according to either clause 67 or 144,         wherein the product saponin is desarabinofuranosyl-QS-21 1870 A         V1.     -   Clause 146. The method according to either clause 68 or 144,         wherein the product saponin is desarabinofuranosyl-QS-21 1870 A         V2.     -   Clause 147. The method according to either clause 69 or 139,         wherein the product saponin is a desarabinofuranosyl-QS-21 1856         B component.     -   Clause 148. The method according to either clause 70 or 147,         wherein the product saponin is desarabinofuranosyl-QS-21 1856 B         V1.     -   Clause 149. The method according to either clause 71 or 147,         wherein the product saponin is desarabinofuranosyl-QS-21 1856 B         V2.     -   Clause 150. The method according to either clause 72 or 139,         wherein the product saponin is desarabinofuranosyl-QS-21 1712 B         component.     -   Clause 151. The method according to either clause 73 or 139,         wherein the product saponin is a desarabinofuranosyl-QS-21 1870         B component.     -   Clause 152. The method according to either clause 74 or 151,         wherein the product saponin is desarabinofuranosyl-QS-21 1870 B         V1.     -   Clause 153. The method according to either clause 75 or 151,         wherein the product saponin is desarabinofuranosyl-QS-21 1870 B         V2.     -   Clause 154. The method according to either clause 76 or 93,         wherein the product saponin is an acetylated QS-21 family         component.     -   Clause 155. The method according to either clause 77 or 154,         wherein the product saponin is an acetylated QS-21 2030 A         component.     -   Clause 156. The method according to either clause 78 or 155,         wherein the product saponin is acetylated QS-21 2030 A V1.     -   Clause 157. The method according to either clause 79 or 155,         wherein the product saponin is acetylated QS-21 2030 A V2.     -   Clause 158. The method according to either clause 80 or 154,         wherein the product saponin is acetylated QS-21 1898 A         component.     -   Clause 159. The method according to either clause 81 or 155,         wherein the product saponin is an acetylated QS-21 2044 A         component.     -   Clause 160. The method according to either clause 82 or 159,         wherein the product saponin is acetylated QS-21 2044 A V1.     -   Clause 161. The method according to either clause 83 or 159,         wherein the product saponin is acetylated QS-21 2044 A V2.     -   Clause 162. The method according to any one of clauses 1 to 161,         wherein a single starting saponin is converted to a single         product saponin.     -   Clause 163. The method according to any one of clauses 1 to 161,         wherein a plurality of starting saponins are converted to a         plurality of product saponins.     -   Clause 164. The method according to clause 163, wherein the         plurality of starting saponins comprises QS-18 family         components, such as described in any one of clauses 18 to 31.     -   Clause 165. The method according to either clause 163 or 164,         wherein the plurality of starting saponins comprises         desglucosyl-QS-17 family components, such as described in any         one of clauses 33 to 46.     -   Clause 166. The method according to any one of clauses 163 to         165, wherein the plurality of starting saponins comprises QS-17         family components, such as described in any one of clauses 48 to         61.     -   Clause 167. The method according to any one of clauses 163 to         166, wherein the plurality of starting saponins comprises         desarabinofuranosyl-QS-18 family components, such as described         in any one of clauses 63 to 76.     -   Clause 169. The method according to any one of clauses 163 to         167, wherein the plurality of starting saponins comprises         acetylated desglucosyl-QS-17 family components, such as         described in any one of clauses 78 to 84.     -   Clause 169. The method according to any one of clauses 1 to 4 or         6 to 168, wherein the starting saponin is obtained by extraction         from starting material.     -   Clause 170. The method according to clause 169, wherein the         starting saponin is obtained by extraction from plant material.     -   Clause 171. The method according to clause 170, wherein the         starting saponin is obtained by extraction from plant material         from plants of the genera Gypsophilia, Saponaria or Quillaja.     -   Clause 172. The method according to clause 171, wherein the         starting saponin is obtained by extraction from plant material         obtained from Quillaja species.     -   Clause 173. The method according to clause 172, wherein the         starting saponin is obtained by extraction from plant material         obtained from Quillaja brasiliensis.     -   Clause 174. The method according to clause 172, wherein the         starting saponin is obtained by extraction from plant material         obtained from Quillaja saponaria.     -   Clause 175. The method according to any one of clauses 169 to         174, wherein the starting saponin is obtained by extraction from         complete plants.     -   Clause 176. The method according to any one of clauses 169 to         174, wherein the starting saponin is obtained by extraction from         selected plant tissues.     -   Clause 177. The method according to clause 176, wherein the         starting saponin is obtained by extraction from plant material         including wood or bark, such as plant material which is wood or         bark.     -   Clause 178. The method according to clause 177, wherein the         starting saponin is obtained by extraction from plant material         including bark, such as plant material which is bark.     -   Clause 179. The method according to any one of clauses 169 to         188, wherein the starting saponin is obtained by extraction from         plant material obtained from an adult plant.     -   Clause 180. The method according to any one of clauses 169 to         179, wherein the starting saponin is obtained by extraction from         plant material obtained from a young plant.     -   Clause 181. The method according to any one of clauses 169 to         180, wherein the starting saponin is obtained by extraction         using water and/or lower alcohols.     -   Clause 182. The method according to clause 181, wherein the         starting saponin is obtained by aqueous extraction.     -   Clause 183. The method according to any one of clauses 1 to 182,         wherein the starting saponin is a minor component in a saponin         containing composition, such as a minor component in a plant         material extract.     -   Clause 184. The method according to any one of clauses 1 to 182,         wherein the starting saponin is a major component in a saponin         containing composition, such as a major component in a plant         material extract.     -   Clause 185. The method according to any one of clauses 169 to         184, wherein the starting saponin is partially purified.     -   Clause 186. The method according to any one of clauses 169 to         184, wherein the starting saponin is substantially purified.     -   Clause 187. The method according to any one of clauses 169 to         184, wherein the starting saponin is in the form of a component         of Quil A.     -   Clause 188. The method according to any one of clauses 169 to         184, wherein the starting saponin is in the form of a component         of Fraction A.     -   Clause 189. The method according to any one of clauses 169 to         184, wherein the starting saponin is in the form of a component         of Fraction B.     -   Clause 190. The method according to any one of clauses 169 to         184, wherein the starting saponin is in the form of a component         of Fraction C.     -   Clause 191. The method according to any one of clauses 169 to         184, wherein the starting saponin is in the form of a component         of QS-7.     -   Clause 192. The method according to any one of clauses 169 to         184, wherein the starting saponin is in the form of a component         of QS-17.     -   Clause 193. The method according to any one of clauses 169 to         184, wherein the starting saponin is in the form of a component         of QS-18.     -   Clause 194. The method according to any one of clauses 169 to         184, wherein the starting saponin is in the form of a component         of QS-21.     -   Clause 195. The method according to any one of clauses 169 to         184, wherein the starting saponin is in the form of a component         of a crude bark extract.     -   Clause 196. The method according to any one of clauses 169 to         195, further comprising the step of extracting the starting         saponin from the starting material.     -   Clause 197. The method according to any one of clauses 1 to 194         or 196, further comprising the step of purifying the starting         saponin from extracted material.     -   Clause 198. The method according to any one of clauses 1 to 197,         wherein the enzymatic conversion involves the removal of a         single sugar residue.     -   Clause 199. The method according to clause 198, wherein the         enzymatic conversion involves the removal of a glucose residue.     -   Clause 200. The method according to clause 199, wherein the         enzymatic conversion involves the removal of a beta-glucose         residue.     -   Clause 201. The method according to clause 200, wherein the         enzymatic conversion involves the removal of the beta-glucose         residue:     -   Clause 202. The method according to clause 198, wherein the         enzymatic conversion involves the removal of a rhamnose residue.     -   Clause 203. The method according to clause 202, wherein the         enzymatic conversion involves the removal of a alpha-rhamnose         residue.     -   Clause 204. The method according to clause 203, wherein the         enzymatic conversion involves the removal of the alpha-rhamnose         residue:     -   Clause 205. The method according to any one of clauses 1 to 197,         wherein the enzymatic conversion involves the removal of a         plurality of sugar residues.     -   Clause 206. The method according to any one of clauses 1 to 205,         wherein the method involves multiple enzymatic conversions.     -   Clause 207. The method according to clause 206, wherein the         multiple enzymatic conversions are undertaken in series.     -   Clause 208. The method according to clause 206, wherein the         multiple enzymatic conversions are undertaken in parallel.     -   Clause 209. The method according to any one of clauses 206 to         208, wherein the multiple enzymatic conversions comprise, such         as consist of, the removal of glucose and rhamnose.     -   Clause 210. The method according to clause 209, wherein the         multiple enzymatic conversions comprise, such as consist of, the         removal of a beta-glucose residue and an alpha-rhamnose residue.     -   Clause 211. The method according to clause 210, wherein the         multiple enzymatic conversions comprise, such as consist of, the         removal of:     -   Clause 212. The method according to any one of clauses 1 to 30,         46 to 75, 84 to 108, 124 to 153 or 162 to 197 wherein the         enzymatic conversion is carried out by an enzyme demonstrating         beta exo glucosidase activity.     -   Clause 213. The method according to any one of clauses 1 to 15,         31 to 45, 76 to 123 or 154 to 197 wherein the enzymatic         conversion is carried out by an enzyme demonstrating alpha exo         rhamnosidase activity.     -   Clause 214. The method according to any one of clauses 1 to 213,         wherein the glycosidase is of external origin.     -   Clause 215. The method according to any one of clauses 1 to 214,         wherein the enzymatic conversion occurs in an extra cellular         environment.     -   Clause 216. The method according to any one of clauses 1 to 215,         wherein the glycosidase is recombinantly produced.     -   Clause 217. The method according to any one of clauses 1 to 216,         wherein the glycosidase is provided in the form of a lysate,         such as a clarified lysate and in particular an E. coli lysate         or clarified lysate.     -   Clause 218. The method according to any one of clauses 1 to 217,         further comprising the step of purifying the product saponin.     -   Clause 219. The method according to any one of clauses 1 to 218,         wherein the enzymatic conversion is undertaken at pH 4 to 9,         especially pH 5 to 8, and in particular pH to 7.5 such as pH 5.5         to 6.5.     -   Clause 220. The method according to any one of clauses 1 to 219,         wherein the enzymatic conversion is undertaken at 10 degC to 60         degC, especially 15 degC to 50 degC, in particular 15 degC to 45         degC, such as 20 degC to 42 degC.     -   Clause 221. The method according to any one of clauses 1 to 220,         wherein the enzymatic conversion occurs over a period of up to 2         days, especially up to 1 day, in particular up to 18 hrs, such         as 12 hrs, for example up to 6 hrs.     -   Clause 222. The method according to any one of clauses 1 to 221,         wherein the enzymatic conversion occurs in water or an aqueous         solution with water miscible co-solvent(s).     -   Clause 223. The method according to any one of clauses 1 to 222,         wherein the starting saponins are present at a concentration of         0.001 to 100 g per litre, especially to 75 g per litre, in         particular 0.01 to 50 g per litre, such as 0.1 to 25 g per         litre, for example 1 to 10 g per litre.     -   Clause 224. The method according to any one of clauses 1 to 223,         wherein the enzymatic conversion occurs in a batch reaction         volume of 500 ml to 2000 L, especially 1 L to 1000 L, in         particular 10 L to 500 L, such as 25 L to 200 L.     -   Clause 225. The method according to any one of clauses 1 to 224,         wherein the weight of each glycosidase present is in the range         of 0.0001 mg to 25 mg per ml, especially 0.0001 mg to 5 mg per         ml, in particular 0.0001 mg to 1 mg per ml, such as 0.001 mg to         0.5 mg per ml.     -   Clause 226. The method according to any one of clauses 1 to 225,         wherein the weight of each glycosidase present is in the range         of 0.01 mg to 100 mg of dried clarified lysate per ml,         especially 0.01 mg to 30 mg per ml, in particular 0.01 mg to 5         mg per ml, such as 0.01 mg to 1 mg per ml.     -   Clause 227. Use of a glycosidase for enzymatically converting a         starting saponin to the product saponin, such as in a method of         any one of clauses 1 to 226.     -   Clause 228. The method or use according to any one of clauses         212 or 214 to 227, wherein the glucosidase comprises, such as         consists of, an amino acid sequence according to SEQ ID No. 262,         208, 63, 229, 250, 5, 101, 207, 169, 247, 302, 324, 319, 9, 240,         325, 338, 850, 879, 868, 826, 804, 888, 881, 891, 816, 827, 857,         853, 842, 814, 886, 885, 838, 829, 808, 828, 870, 873, 844, 882,         874, 825, 824, 823, 810, 894, 849, 803, 890, 841, 832, 830, 845,         871, 837, 883 or 809 or functional variants thereof.     -   Clause 229. A method for identifying a candidate enzyme having         beta exo glucosidase activity, comprising selecting an enzyme         comprising, such as consisting of, an amino acid sequence         according to SEQ ID No. 262, 208, 63, 229, 250, 5, 101, 207,         169, 247, 302, 324, 319, 9, 240, 325, 338, 850, 879, 868, 826,         804, 888, 881, 891, 816, 827, 857, 853, 842, 814, 886, 885, 838,         829, 808, 828, 870, 873, 844, 882, 874, 825, 824, 823, 810, 894,         849, 803, 890, 841, 832, 830, 845, 871, 837, 883 or 809 or         functional variants thereof.     -   Clause 230. A method for identifying an enzyme having beta exo         glucosidase activity, comprising testing a glycosidase, such as         an enzyme comprising, such as consisting of, an amino acid         sequence according to SEQ ID No. 262, 208, 63, 229, 250, 5, 101,         207, 169, 247, 302, 324, 319, 9, 240, 325, 338, 850, 879, 868,         826, 804, 888, 881, 891, 816, 827, 857, 853, 842, 814, 886, 885,         838, 829, 808, 828, 870, 873, 844, 882, 874, 825, 824, 823, 810,         894, 849, 803, 890, 841, 832, 830, 845, 871, 837, 883 or 809 or         functional variants thereof, for ability to remove glucose from         a saponin.     -   Clause 231. The use or method according to any one of clauses         227 to 230, wherein the enzyme comprises, such as consists of,         an amino acid sequence according to SEQ ID No. 262, 208, 63,         229, 250, 5, 101, 207, 169, 247, 302, 324, 319, 9, 240, 325 or         338, or functional variants thereof.     -   Clause 232. The use or method according to clause 231, wherein         the enzyme comprises, such as consists of, an amino acid         sequence according to SEQ ID No. 262, 208, 63, 229, 250, 5, 101,         207, 169, 247, 302, 324 or 319, or functional variants thereof.     -   Clause 233. The use or method according to clause 232, wherein         the enzyme comprises, such as consists of, an amino acid         sequence according to SEQ ID No. 262, 208, 63, 229, 250, 5, 101         or 207, or functional variants thereof.     -   Clause 234. The use or method according to clause 233, wherein         the enzyme comprises, such as consists of, an amino acid         sequence according to SEQ ID No. 262, or functional variants         thereof.     -   Clause 235. The use or method according to clause 234, wherein         the enzyme is an engineered glucosidase polypeptide according to         any one of clauses 260 to 355.     -   Clause 236. The use or method according to any one of clauses         227 to 230, wherein the enzyme comprises, such as consists of,         an amino acid sequence according to SEQ ID No. 850, 879, 868,         826, 804, 888, 881, 891, 816, 827, 857, 853, 842, 814, 886, 885,         838, 829, 808, 828, 870, 873, 844, 882, 874, 825, 824, 823, 810,         894, 849, 803, 890, 841, 832, 830, 845, 871, 837, 883 or 809, or         functional variants thereof.     -   Clause 237. The use or method according to clause 236, wherein         the enzyme comprises, such as consists of, an amino acid         sequence according to SEQ ID No. 850, 879, 868, 826, 804, 888,         881, 891, 816, 827, 857, 853, 842, 814, 886, 885, 838, 829, 808,         828, 870, 873, 844, 882, 874, 825, 824, 823, 810, 894, 849, 803,         890 or 841, or functional variants thereof.     -   Clause 238. The use or method according to clause 237, wherein         the enzyme comprises, such as consists of, an amino acid         sequence according to SEQ ID No. 850, 879, 868, 826, 804, 888,         881, 891, 816, 827, 857, 853, 842, 814, 886, 885, 838, 829, 808,         828, 870, 873, 844, 882, 874, 825, 824, 823, 810 or 894, or         functional variants thereof.     -   Clause 239. The method or use according to any one of clauses         213 to 227, wherein the rhamnosidase comprises, such as consists         of, an amino acid sequence according to SEQ ID No. 992, 1003,         1052, 1073, 1017, 1055, 1075, 1001, 1007, 1061, 1079, 1027,         1039, 1041, 989, 1053, 1018, 1066, 1082, 1076, 993, 1077, 1046,         1015, 1063, 1054, 1074, 1067 or 1033, or functional variants         thereof.     -   Clause 240. A method for identifying a candidate enzyme having         alpha exo rhamnosidase activity, comprising selecting an enzyme         comprising, such as consisting of, an amino acid sequence         according to SEQ ID No. 992, 1003, 1052, 1073, 1017, 1055, 1075,         1001, 1007, 1061, 1079, 1027, 1039, 1041, 989, 1053, 1018, 1066,         1082, 1076, 993, 1077, 1046, 1015, 1063, 1054, 1074, 1067 or         1033, or functional variants thereof.     -   Clause 241. A method for identifying an enzyme having alpha exo         rhamnosidase activity, comprising testing a glycosidase, such as         enzyme comprising, such as consisting of, an amino acid sequence         according to SEQ ID No. 992, 1003, 1052, 1073, 1017, 1055, 1075,         1001, 1007, 1061, 1079, 1027, 1039, 1041, 989, 1053, 1018, 1066,         1082, 1076, 993, 1077, 1046, 1015, 1063, 1054, 1074, 1067 or         1033, or functional variants thereof for ability to remove         rhamnose from a saponin.     -   Clause 242. The use or method according to any one of clauses         239 to 241, wherein the enzyme comprises, such as consists of,         an amino acid sequence according to SEQ ID No. 992, 1003, 1052,         1073, 1017, 1055, 1075, 1001, 1007, 1061, 1079, 1027, 1039,         1041, 989, 1053, 1018, 1066, 1082, 1076, 993 or 1077, or         functional variants thereof.     -   Clause 243. The use or method according to clause 242, wherein         the enzyme comprises, such as consists of, an amino acid         sequence according to SEQ ID No. 992, 1003, 1052, 1073, 1017,         1055, 1075, 1001, 1007, 1061, 1079, 1027, 1039, 1041 or 989, or         functional variants thereof.     -   Clause 244. The use or method according to clause 243, wherein         the enzyme comprises, such as consists of, an amino acid         sequence according to SEQ ID No. 1017, or functional variants         thereof.     -   Clause 245. The use or method according to clause 243, wherein         the enzyme is an engineered rhamnosidase polypeptide according         to any one of clauses 376 to 463.     -   Clause 246. The use or method according to any one of clauses         228 to 245, wherein the functional variant comprises a sequence         having at least 80% identity to the reference sequence,         especially at least 90%, in particular at least 95%, such as at         least 98%, for example at least 99% identity.     -   Clause 247. The use or method according to any one of clauses         228 to 245, wherein the functional variant comprises a sequence         having a fragment of at least at least 100, especially at least         200, particularly at least 300, such as at least 400, for         example at least 500 contiguous amino acids of the reference         sequence.     -   Clause 248. The use or method according to any one of clauses         228 to 245, wherein the functional variant comprises a sequence         having 1 to 20 additions, deletions and/or substitutions         relative to the reference sequence, especially 1 to 15         additions, deletions and/or substitutions, particularly 1 to 10         additions, deletions and/or substitutions, such as 1 to 5         additions, deletions and/or substitutions.     -   Clause 249. The use or method according to any one of clauses         228 to 245, wherein the enzyme comprises, such as consists of, a         reference sequence.     -   Clause 250. A saponin prepared by the method of any one of         clauses 1 to 226, 228, 231 to 239 or 242 to 249.     -   Clause 251. A saponin containing composition comprising a         product saponin prepared by the method of any one of clauses 1         to 226, 228, 231 to 239 or 242 to 249.     -   Clause 252. The saponin containing composition according to         clause 251, comprising QS-21 family components.     -   Clause 253. An adjuvant composition comprising a saponin or         saponin containing composition according to any one of clauses         250 to 252.     -   Clause 254. An adjuvant composition prepared using a saponin or         saponin containing composition according to any one of clauses         250 to 252.     -   Clause 255. Use of a saponin or saponin containing composition         according to any one of clauses 250 to 252 in the manufacture of         an adjuvant composition.     -   Clause 256. An immunogenic composition comprising a saponin or         saponin containing composition according to any one of clauses         250 to 252 and an antigen.     -   Clause 257. An immunogenic composition comprising a saponin or         saponin containing composition according to any one of clauses         250 to 252 and a polynucleotide encoding an antigen.     -   Clause 258. A kit of parts comprising:         -   (i) a saponin or saponin containing composition according to             any one of clauses 250 to 252         -   (ii) an antigen.     -   Clause 259. A kit of parts comprising:         -   (i) a saponin or saponin composition according to any one of             clauses 250 to 252         -   (ii) a polynucleotide encoding an antigen.     -   Clause 260. An engineered glucosidase polypeptide comprising an         amino acid sequence that is at least 80% identical to the amino         acid sequence of SEQ ID No. 262, or a functional fragment         thereof, wherein the engineered glucosidase polypeptide includes         at least one residue substitution from:         -   F44Y;         -   V60L;         -   G117A;         -   F170N;         -   V263G or V263L;         -   N351H or N351Q;         -   A355H, A355I, A355L, A355M, A355R, A355T or A355W;         -   A356P;         -   R357A, R357C, R357K, R357M or R357Q;         -   G362C;         -   T365A, T365N or T365S;         -   L367C;         -   V394R;         -   V395Y;         -   Q396E, Q396G, Q396N, Q396P, Q396R, Q396S or Q396Y;         -   F430W;         -   R435F;         -   V438T;         -   V440F;         -   F442M or F442Q;         -   G444T;         -   A473F or A473R;         -   L474C, L474I or L474V;         -   I475F;         -   L492C, L492G, L492H, L492I, L492N, L492Q, L492V, L492W or             L492Y;         -   Q493F or Q493H;         -   P494H or P494I;         -   S495I, S495K or S495Q;         -   G496P or G496W;         -   D498A, D498E, D498F, D498I, D498K, D498L, D498N, D498P,             D498R, D498S, D498T or D498V;         -   A502R;         -   M504G or M504R;         -   L507A or L507R;         -   T508M;         -   L529M;         -   F535P;         -   A536D or A536E;         -   A537R;         -   F541A, F541I, F541L, F541M or F541V;         -   L542I;         -   Q543G or Q543L;         -   E547L; and         -   Y585W.     -   Clause 261. The polypeptide according to clause 260, having one         of the substitutions.     -   Clause 262. The polypeptide according to clause 260, having two         of the substitutions.     -   Clause 263. The polypeptide according to clause 260, having         three of the substitutions.     -   Clause 264. The polypeptide according to clause 260, having four         of the substitutions.     -   Clause 265. The polypeptide according to clause 260, having five         of the substitutions.     -   Clause 266. The polypeptide according to clause 260, having six         of the substitutions.     -   Clause 267. The polypeptide according to clause 260, having         seven of the substitutions.     -   Clause 268. The polypeptide according to clause 260, having         eight of the substitutions.     -   Clause 269. The polypeptide according to clause 260, having nine         of the substitutions.     -   Clause 270. The polypeptide according to clause 260, having ten         of the substitutions.     -   Clause 271. The polypeptide according to clause 260, having         eleven of the substitutions.     -   Clause 272. The polypeptide according to clause 260, having         twelve of the substitutions.     -   Clause 273. The polypeptide according to clause 260, having         thirteen of the substitutions.     -   Clause 274. The polypeptide according to clause 260, having         fourteen of the substitutions.     -   Clause 275. The polypeptide according to clause 260, having         fifteen of the substitutions.     -   Clause 276. The polypeptide according to clause 260, having         sixteen of the substitutions.     -   Clause 277. The polypeptide according to clause 260, having         seventeen of the substitutions.     -   Clause 277. The polypeptide according to clause 260, having         eighteen of the substitutions.     -   Clause 278. The polypeptide according to clause 260, having         nineteen of the substitutions.     -   Clause 279. The polypeptide according to clause 260, having         twenty of the substitutions.     -   Clause 280. The polypeptide according to clause 260, having         twenty-one of the substitutions.     -   Clause 281. The polypeptide according to clause 260, having         twenty-two of the substitutions.     -   Clause 282. The polypeptide according to clause 260, having         twenty-three of the substitutions.     -   Clause 283. The polypeptide according to clause 260, having         twenty-four of the substitutions.     -   Clause 284. The polypeptide according to clause 260, having         twenty-five of the substitutions.     -   Clause 285. The polypeptide according to clause 260, having         twenty-six to thirty of the substitutions.     -   Clause 286. The polypeptide according to clause 260, having         thirty-one to forty-three substitutions of the substitutions.     -   Clause 287. The polypeptide according to any one of clauses 260         to 286, comprising F44Y.     -   Clause 288. The polypeptide according to any one of clauses 260         to 287, comprising V60L.     -   Clause 289. The polypeptide according to any one of clauses 260         to 288, comprising G117A.     -   Clause 290. The polypeptide according to any one of clauses 260         to 289, comprising F170N.     -   Clause 291. The polypeptide according to any one of clauses 260         to 290, comprising V263G or V263L.     -   Clause 292. The polypeptide according to clause 291, comprising         V263L.     -   Clause 293. The polypeptide according to any one of clauses 260         to 292, comprising N351H or N351Q.     -   Clause 294. The polypeptide according to clause 293, comprising         N351H.     -   Clause 295. The polypeptide according to any one of clauses 260         to 294, comprising A355H, A355I, A355L, A355M, A355R, A355T or         A355W.     -   Clause 296. The polypeptide according to clause 295, comprising         A355H.     -   Clause 297. The polypeptide according to clause 295, comprising         A355I.     -   Clause 298. The polypeptide according to clause 295, comprising         A355L.     -   Clause 299. The polypeptide according to clause 295, comprising         A355M.     -   Clause 300. The polypeptide according to clause 295, comprising         A355R.     -   Clause 301. The polypeptide according to clause 295, comprising         A355T.     -   Clause 302. The polypeptide according to clause 295, comprising         A355W.     -   Clause 303. The polypeptide according to any one of clauses 260         to 302, comprising A356P.     -   Clause 304. The polypeptide according to any one of clauses 260         to 303, comprising R357A, R357C, R357K, R357M or R357Q.     -   Clause 305. The polypeptide according to clause 304, comprising         R357M.     -   Clause 306. The polypeptide according to any one of clauses 260         to 305, comprising G362C.     -   Clause 307. The polypeptide according to any one of clauses 260         to 306, comprising T365A, T365N or T365S.     -   Clause 308. The polypeptide according to clause 307, comprising         T365N.     -   Clause 309. The polypeptide according to any one of clauses 260         to 308, comprising L367C.     -   Clause 310. The polypeptide according to any one of clauses 260         to 309, comprising V394R.     -   Clause 311. The polypeptide according to any one of clauses 260         to 310, comprising V395Y.     -   Clause 312. The polypeptide according to any one of clauses 260         to 311, comprising Q396E, Q396G, Q396N, Q396P, Q396R, Q396S or         Q396Y.     -   Clause 313. The polypeptide according to clause 312, comprising         Q396R.     -   Clause 314. The polypeptide according to any one of clauses 260         to 313, comprising F430W.     -   Clause 315. The polypeptide according to any one of clauses 260         to 314, comprising R435F.     -   Clause 316. The polypeptide according to any one of clauses 260         to 315, comprising V438T.     -   Clause 317. The polypeptide according to any one of clauses 260         to 316, comprising V440F.     -   Clause 318. The polypeptide according to any one of clauses 260         to 317, comprising F442M or F442Q.     -   Clause 319. The polypeptide according to clause 318, comprising         F442Q.     -   Clause 320. The polypeptide according to any one of clauses 260         to 319, comprising G444T.     -   Clause 321. The polypeptide according to any one of clauses 260         to 320, comprising A473F or A473R.     -   Clause 322. The polypeptide according to clause 321, comprising         A473F.     -   Clause 323. The polypeptide according to any one of clauses 260         to 322, comprising L474C, L474I or L474V.     -   Clause 324. The polypeptide according to clause 323, comprising         L474C.     -   Clause 325. The polypeptide according to any one of clauses 260         to 324, comprising I475F.     -   Clause 326. The polypeptide according to any one of clauses 260         to 325, comprising L492C, L492G, L492H, L492I, L492N, L492Q,         L492V, L492W or L492Y.     -   Clause 327. The polypeptide according to clause 326, comprising         L492H.     -   Clause 328. The polypeptide according to clause 326, comprising         L492N.     -   Clause 329. The polypeptide according to clause 326, comprising         L492V.     -   Clause 330. The polypeptide according to any one of clauses 260         to 329, comprising Q493F or Q493H.     -   Clause 331. The polypeptide according to any one of clauses 260         to 330, comprising P494I.     -   Clause 332. The polypeptide according to any one of clauses 260         to 331, comprising S495I, S495K or S495Q.     -   Clause 333. The polypeptide according to any one of clauses 260         to 332, comprising G496P or G496W.     -   Clause 334. The polypeptide according to any one of clauses 260         to 333, comprising G496P.     -   Clause 335. The polypeptide according to any one of clauses 260         to 334, comprising D498A, D498E, D498F, D498I, D498K, D498L,         D498N, D498P, D498R, D498S, D498T or D498V.     -   Clause 336. The polypeptide according to clause 335, comprising         D498P.     -   Clause 337. The polypeptide according to any one of clauses 260         to 336, comprising A502R;     -   Clause 338. The polypeptide according to any one of clauses 260         to 337, comprising M504G or M504R.     -   Clause 339. The polypeptide according to any one of clauses 260         to 338, comprising M504R.     -   Clause 340. The polypeptide according to any one of clauses 260         to 339, comprising L507A or L507R.     -   Clause 341. The polypeptide according to clause 340, comprising         L507R.     -   Clause 342. The polypeptide according to any one of clauses 260         to 341, comprising T508M.     -   Clause 343. The polypeptide according to any one of clauses 260         to 342, comprising L529M.     -   Clause 344. The polypeptide according to any one of clauses 260         to 343, comprising F535P.     -   Clause 345. The polypeptide according to any one of clauses 260         to 344, comprising A536D or A536E.     -   Clause 346. The polypeptide according to any one of clauses 260         to 345, comprising A537R.     -   Clause 347. The polypeptide according to any one of clauses 260         to 346, comprising F541A, F541I, F541L, F541M or F541V.     -   Clause 348. The polypeptide according to clause 347, comprising         F541I.     -   Clause 349. The polypeptide according to any one of clauses 260         to 348, comprising L542I.     -   Clause 350. The polypeptide according to any one of clauses 260         to 349, comprising Q543G or Q543L.     -   Clause 351. The polypeptide according to any one of clauses 260         to 350, comprising E547L.     -   Clause 352. The polypeptide according to any one of clauses 260         to 351, comprising Y585W.     -   Clause 353. The polypeptide according to clause 260, comprising         one mutation which is T365N.     -   Clause 354. The polypeptide according to clause 260 or 265 to         353, comprising R357M, T365N, A473F, L474C and I475F.     -   Clause 355. The polypeptide according to clause 260 or 267 to         353, comprising F44Y, R357M, T365N, F442Q, A473F, L474C and         I475F.     -   Clause 356. The polypeptide according to clause 260 or 269 to         353, comprising F44Y, V263L, R357M, T365N, F442Q, A473F, L474C,         I475F and F541I.     -   Clause 357. The polypeptide according to clause 260 to 353,         comprising at least one residue substitution from F44Y, V263L,         A355W, R357M, T365N, L367C, Q396R, F442Q, L474C, I475F and         F541I,     -   Clause 358. The polypeptide according to clause 357, comprising         F44Y, V263L, A355W, R357M, T365N, L367C, Q396R, F442Q, L474C,         1475F and F541I,     -   Clause 359. The polypeptide according to any one of clauses 260         to 358, comprising an amino acid sequence that is at least 80%         identical to the amino acid sequence of SEQ ID No. 262, or a         functional fragment thereof, wherein the engineered glucosidase         polypeptide includes at least one residue substitution from:         -   F44Y;         -   V263L;         -   N351H;         -   A355H, A355M or A355W;         -   R357M;         -   T365N;         -   L367C;         -   Q396R;         -   V438T;         -   F442Q;         -   L474C;         -   I475F;         -   L492V, L492N or L492H,         -   M504R;         -   L507R; and         -   F541I.     -   Clause 359. The polypeptide according to any one of clauses 260         to 358 comprising an amino acid sequence that is at least 85%         identical to the amino acid sequence of SEQ ID No. 262.     -   Clause 360. The polypeptide according to any one of clauses 260         to 358 comprising an amino acid sequence that is at least 90%         identical to the amino acid sequence of SEQ ID No. 262.     -   Clause 361. The polypeptide according to any one of clauses 260         to 358 comprising an amino acid sequence that is at least 95%         identical to the amino acid sequence of SEQ ID No. 262.     -   Clause 362. The polypeptide according to any one of clauses 260         to 358 comprising an amino acid sequence that is at least 98%         identical to the amino acid sequence of SEQ ID No. 262.     -   Clause 363. The polypeptide according to any one of clauses 260         to 358, wherein the functional variant comprises a sequence         having a fragment of at least 100, especially at least 200,         particularly at least 300, such as at least 400, for example at         least 500 contiguous amino acids of SEQ ID No. 262.     -   Clause 364. The polypeptide according to clause 260, comprising         the amino acid sequence of SEQ ID No. 1179.     -   Clause 365. The polypeptide according to clause 260, comprising         the amino acid sequence of SEQ ID No. 1180.     -   Clause 366. The polypeptide according to clause 260, comprising         the amino acid sequence of SEQ ID No. 1181.     -   Clause 367. The polypeptide according to clause 260, comprising         the amino acid sequence of SEQ ID No. 1182.     -   Clause 368. The polypeptide according to clause 260, comprising         the amino acid sequence of SEQ ID No. 1183.     -   Clause 369. The polypeptide according to any one of clauses 260         to 368, further comprising an affinity tag.     -   Clause 370. The polypeptide according to clause 369, wherein the         affinity tag is a poly-his tag, such as a hexa-his tag.     -   Clause 371. The polypeptide according to either clause 369 or         370, wherein the affinity tag is N-terminally located.     -   Clause 372. The polypeptide according to clause 371, comprising         an amino acid sequence of SEQ ID No. 1177.     -   Clause 373. The polypeptide according to either clause 369 or         370, wherein the affinity tag is C-terminally located.     -   Clause 374. The polypeptide according to clause 373, comprising         an amino acid sequence of SEQ ID No. 1178.     -   Clause 375. The polypeptide according to any one of clauses 260         to 374, comprising 1000 residues or fewer, especially 950         residues or fewer, in particular 900 residues or fewer, such as         850 residues or fewer.     -   Clause 376. An engineered rhamnosidase polypeptide comprising an         amino acid sequence that is at least 80% identical to the amino         acid sequence of SEQ ID No. 1017, or a functional fragment         thereof, wherein the engineered rhamnosidase polypeptide         includes at least one residue substitution from:         -   (i) A56C         -   (ii) A143P         -   (iii) Q181H, Q181R or Q181S         -   (v) G215S         -   (vi) F216M         -   (vii) G218D or G218N         -   (viii) K219G         -   (ix) A238M         -   (x) T252Y         -   (xi) T311W         -   (xii) V326C         -   (xiii) G357C         -   (xiv) S369C, S369I, S369K or S369M         -   (xv) I487M, I487Q or I487V         -   (xvi) K492N         -   (xvii) V499T         -   (xviii) G508S         -   (xix) R543C         -   (xx) L557Y         -   (xxi) G634A         -   (xxii) S635N         -   (xxiii) A690C and         -   (xxiv) Q921H.     -   Clause 377. A polypeptide comprising an amino acid sequence of         sequence of SEQ ID No. 1017 with one to twenty-four mutations         selected from the list consisting of:         -   (i) A56C         -   (ii) A143P         -   (iii) Q181H, Q181R or Q181S         -   (iv) L214M         -   (v) G215S         -   (vi) F216M         -   (vii) G218D or G218N         -   (viii) K219G         -   (ix) A238M         -   (x) T252Y         -   (xi) T311W         -   (xii) V326C         -   (xiii) G357C         -   (xiv) S369C, S369I, S369K or S369M         -   (xv) I487M, I487Q or I487V         -   (xvi) K492N         -   (xvii) V499T         -   (xviii) G508S         -   (xix) R543C         -   (xx) L557Y         -   (xxi) G634A         -   (xxii) S635N         -   (xxiii) A690C and         -   (xxiv) Q921H.     -   Clause 378. The polypeptide according to either clause 376 or         377, having one of the substitutions.     -   Clause 379. The polypeptide according to either clause 376 or         377, having two of the substitutions.     -   Clause 380. The polypeptide according to either clause 376 or         377, having three of the substitutions.     -   Clause 381. The polypeptide according to either clause 376 or         377, having four of the substitutions.     -   Clause 382. The polypeptide according to either clause 376 or         377, having five of the substitutions.     -   Clause 383. The polypeptide according to either clause 376 or         377, having six of the substitutions.     -   Clause 384. The polypeptide according to either clause 376 or         377, having seven of the substitutions.     -   Clause 385. The polypeptide according to either clause 376 or         377, having eight of the substitutions.     -   Clause 386. The polypeptide according to either clause 376 or         377, having nine of the substitutions.     -   Clause 387. The polypeptide according to either clause 376 or         377, having ten of the substitutions.     -   Clause 388. The polypeptide according to either clause 376 or         377, having eleven of the substitutions.     -   Clause 389. The polypeptide according to either clause 376 or         377, having twelve of the substitutions.     -   Clause 390. The polypeptide according to either clause 376 or         377, having thirteen of the substitutions.     -   Clause 391. The polypeptide according to either clause 376 or         377, having fourteen of the substitutions.     -   Clause 392. The polypeptide according to either clause 376 or         377, having fifteen of the substitutions.     -   Clause 393. The polypeptide according to either clause 376 or         377, having sixteen of the substitutions.     -   Clause 394. The polypeptide according to either clause 376 or         377, having seventeen of the substitutions.     -   Clause 395. The polypeptide according to either clause 376 or         377, having eighteen of the substitutions.     -   Clause 396. The polypeptide according to either clause 376 or         377, having nineteen of the substitutions.     -   Clause 397. The polypeptide according to either clause 376 or         377, having twenty of the substitutions.     -   Clause 398. The polypeptide according to either clause 376 or         377, having twenty-one of the substitutions.     -   Clause 399. The polypeptide according to either clause 376 or         377, having twenty-two of the substitutions.     -   Clause 400. The polypeptide according to either clause 376 or         377, having twenty-three of the substitutions.     -   Clause 401. The polypeptide according to either clause 376 or         377, having twenty-four of the substitutions.     -   Clause 402. The polypeptide according to any one of clauses 376         to 401, comprising A56C.     -   Clause 403. The polypeptide according to any one of clauses 376         to 402, comprising A143P.     -   Clause 404. The polypeptide according to any one of clauses 376         to 403, comprising Q181H, Q181R or Q181S.     -   Clause 405. The polypeptide according to clause 404, comprising         Q181H.     -   Clause 406. The polypeptide according to clause 404, comprising         Q181R.     -   Clause 407. The polypeptide according to clause 404, comprising         Q181S.     -   Clause 408. The polypeptide according to any one of clauses 376         to 407, comprising L214M.     -   Clause 409. The polypeptide according to any one of clauses 376         to 408, comprising G215S.     -   Clause 410. The polypeptide according to any one of clauses 376         to 409, comprising F216M.     -   Clause 411. The polypeptide according to any one of clauses 376         to 410, comprising G218D or G218N.     -   Clause 412. The polypeptide according to clause 411, comprising         G218D.     -   Clause 413. The polypeptide according to clause 411, comprising         G218N.     -   Clause 414. The polypeptide according to any one of clauses 376         to 413, comprising K219G.     -   Clause 415. The polypeptide according to any one of clauses 376         to 414, comprising A238M.     -   Clause 416. The polypeptide according to any one of clauses 376         to 415, comprising T252Y.     -   Clause 417. The polypeptide according to any one of clauses 376         to 416, comprising T311W.     -   Clause 418. The polypeptide according to any one of clauses 376         to 417, comprising V326C.     -   Clause 419. The polypeptide according to any one of clauses 376         to 418, comprising G357C.     -   Clause 420. The polypeptide according to any one of clauses 376         to 419, comprising S369C, S369I, S369K or S369M.     -   Clause 421. The polypeptide according to clause 420, comprising         S369C.     -   Clause 422. The polypeptide according to clause 420, comprising         S369I.     -   Clause 423. The polypeptide according to clause 420, comprising         S369K.     -   Clause 424. The polypeptide according to clause 420, comprising         S369M.     -   Clause 425. The polypeptide according to any one of clauses 376         to 424, comprising I487M, I487Q or I487V.     -   Clause 426. The polypeptide according to clause 424, comprising         I487M.     -   Clause 427. The polypeptide according to clause 424, comprising         I487Q.     -   Clause 428. The polypeptide according to clause 424, comprising         I487V.     -   Clause 429. The polypeptide according to any one of clauses 376         to 428, comprising K492N.     -   Clause 430. The polypeptide according to any one of clauses 376         to 429, comprising V499T.     -   Clause 431. The polypeptide according to any one of clauses 376         to 430, comprising G508S.     -   Clause 432. The polypeptide according to any one of clauses 376         to 431, comprising R543C.     -   Clause 433. The polypeptide according to any one of clauses 376         to 432, comprising L557Y.     -   Clause 434. The polypeptide according to any one of clauses 376         to 433, comprising G634A.     -   Clause 435. The polypeptide according to any one of clauses 376         to 434, comprising S635N.     -   Clause 436. The polypeptide according to any one of clauses 376         to 435, comprising A690C.     -   Clause 437. The polypeptide according to any one of clauses 376         to 436, comprising Q921H.     -   Clause 438. The polypeptide according to either clause 376 or         377, comprising one mutation which is K219G.     -   Clause 439. The polypeptide according to either clause 376, 377         or 381 to 401, comprising A143P, L214M, K219G and Q921H.     -   Clause 440. The polypeptide according to clause 376, 377 or 382         to 401, comprising A143P, L214M, K219G, G357C and Q921H.     -   Clause 441. The polypeptide according to clause 376, 377 or 386         to 401, comprising A143P, L214M, G215S, G218N, K219G, G357C,         G508S, G634A and Q921H.     -   Clause 442. The polypeptide according to clause 376 to 401,         comprising at least one residue substitution from A143P, L214M,         G215S, G218D, K219G, G357C, G508S, G634A, A690C and Q921H.     -   Clause 443. The polypeptide according to clause 442, comprising         A143P, L214M, G215S, G218D, K219G, G357C, G508S, G634A, A690C         and Q921H.     -   Clause 444. The polypeptide according to clause 376, 377 or 385         to 401 comprising A143P, L214M, G215S, K219G, G357C, G508S,         G634A and Q921H and one to sixteen mutations selected from the         list consisting of:         -   (i) A56C         -   (iii) Q181H, Q181R or Q181S         -   (vi) F216M         -   (vii) G218D or G218N         -   (ix) A238M         -   (x) T252Y         -   (xi) T311W         -   (xii) V326C         -   (xiv) S369C, S369I, S369K or S369M         -   (xv) I487M, I487Q or I487V         -   (xvi) K492N         -   (xvii) V499T         -   (xix) R543C         -   (xx) L557Y         -   (xxii) S635N and         -   (xxiii) A690C.     -   Clause 445. The polypeptide according to clause 444, comprising         G218D or G218N.     -   Clause 446. The polypeptide according to any one of clauses 376         to 445 comprising an amino acid sequence that is at least 85%         identical to the amino acid sequence of SEQ ID No. 1017.     -   Clause 447. The polypeptide according to any one of clauses 376         to 445 comprising an amino acid sequence that is at least 90%         identical to the amino acid sequence of SEQ ID No. 1017.     -   Clause 448. The polypeptide according to any one of clauses 376         to 445 comprising an amino acid sequence that is at least 95%         identical to the amino acid sequence of SEQ ID No. 1017.     -   Clause 449. The polypeptide according to any one of clauses 376         to 445 comprising an amino acid sequence that is at least 98%         identical to the amino acid sequence of SEQ ID No. 1017.     -   Clause 450. The polypeptide according to any one of clauses 376         to 445, wherein the functional variant comprises a sequence         having a fragment of at least 100, especially at least 200,         particularly at least 300, such as at least 400, for example at         least 500 contiguous amino acids of SEQ ID No. 1017.     -   Clause 451. The polypeptide according to clause 376, comprising         the amino acid sequence of SEQ ID No. 1189.     -   Clause 452. The polypeptide according to clause 376, comprising         the amino acid sequence of SEQ ID No. 1190.     -   Clause 453. The polypeptide according to clause 376, comprising         the amino acid sequence of SEQ ID No. 1191.     -   Clause 454. The polypeptide according to clause 376, comprising         the amino acid sequence of SEQ ID No. 1192.     -   Clause 455. The polypeptide according to clause 376, comprising         the amino acid sequence of SEQ ID No. 1193.     -   Clause 456. The polypeptide according to any one of clauses 376         to 455, further comprising an affinity tag.     -   Clause 457. The polypeptide according to clause 456, wherein the         affinity tag is a poly-his tag, such as a hexa-his tag.     -   Clause 458. The polypeptide according to either clause 456 or         457, wherein the affinity tag is N-terminally located.     -   Clause 459. The polypeptide according to clause 458, comprising         an amino acid sequence of SEQ ID No. 1177.     -   Clause 460. The polypeptide according to either clause 456 or         457, wherein the affinity tag is C-terminally located.     -   Clause 461. The polypeptide according to clause 460, comprising         an amino acid sequence of SEQ ID No. 1178.     -   Clause 462. The polypeptide according to any one of clauses 376         to 461, comprising 1100 residues or fewer, especially 1050         residues or fewer, in particular 1000 residues or fewer, such as         950 residues or fewer.     -   Clause 463. The polypeptide according to any one of clauses 376         to 462, consisting of an amino acid sequence of SEQ ID No. 1         with one to twenty-four mutations selected from the list         consisting of: A56C         -   (ii) A143P         -   (iii) Q181H, Q181R or Q181S         -   (iv) L214M         -   (v) G215S         -   (vi) F216M         -   (vii) G218D or G218N         -   (viii) K219G         -   (ix) A238M         -   (x) T252Y         -   (xi) T311W         -   (xii) V326C         -   (xiii) G357C         -   (xiv) S369C, S369I, S369K or S369M         -   (xv) I487M, I487Q or I487V         -   (xvi) K492N         -   (xvii) V499T         -   (xviii) G508S         -   (xix) R543C         -   (xx) L557Y         -   (xxi) G634A         -   (xxii) S635N         -   (xxiii) A690C and         -   (xxiv) Q921H.     -   Clause 464. A polynucleotide comprising a sequence encoding an         engineered glucosidase or engineered rhamnosidase polypeptide         according to any one of clauses 260 to 463.

The teaching of all references in the present application, including patent applications and granted patents, are herein fully incorporated by reference to the fullest extent possible. A composition or method or process defined as “comprising” certain elements is understood to encompass a composition, method or process (respectively) consisting of those elements. As used herein, ‘consisting essentially of’ means additional components may be present provided they do not alter the overall properties or function.

In respect of numerical values, the terms ‘approximately’, ‘around’ or ‘about’ will typically mean a value within plus or minus 10 percent of the stated value, especially within plus or minus 5 percent of the stated value and in particular the stated value.

Throughout the specification, including the claims, where the context permits, the term “comprising” and variants thereof such as “comprises” are to be interpreted as including the stated element (e.g., integer) or elements (e.g., integers) without necessarily excluding any other elements (e.g., integers). Thus a composition “comprising” X may consist exclusively of X or may include something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.

As used herein, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise.

As used herein, ng refers to nanograms, ug or μg refers to micrograms, mg refers to milligrams, mL or ml refers to milliliter, and mM refers to millimolar. Similar terms, such as um, are to be construed accordingly.

Unless specifically stated, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.

The invention will be further described by reference to the following, non-limiting, examples:

EXAMPLES Example 1—HPLC of a Crude Aqueous Extract of Quillaja saponaria

Crude bark extract was separated by reverse phase HPLC using a C4 column and gradient elution: mobile phase A—water/acetonitrile, 7/3 v/v with 0.15% trifluoroacetic acid; mobile phase B—acetonitrile with 0.15% trifluoroacetic acid. UV detection was at 214 nm.

Crude bark extract samples are diluted as necessary with purified water. Polyvinylpolypyrrolidone (PVPP; 60 mg/mL) was added, the mixture stirred for approximately 30 minutes, and then centrifuged to separate the PVPP resin from the supernatant.

The supernatant was then analysed to provide an HPLC UV chromatogram.

FIG. 1 provides a representative example of an HPLC UV chromatogram. The peak corresponding to the QS-21 fraction is indicated.

Example 2—Analytical Methods HPLC-UV Equipment

-   -   Waters Alliance 2690/2695 separations module     -   Waters 2487 UV Detector or 2996 PDA Detector     -   Vydac Protein C4 4.6×250 mm 5 um column     -   Mobile Phase A (MPA)—0.15% trifluoroacetic acid in         water/acetonitrile (70:30 v/v)     -   Mobile Phase B (MPB)—0.15% trifluoroacetic acid in acetonitrile

TABLE 1 HPLC-UV Linear gradient conditions Time Flow rate (ml/min) % MPA % MPB  0 1 100 0 30 1 78.6 21.4 33 1 14.3 85.7

40 ul of sample is injected. UV detection is set at 214 nM.

Using a blank injection for reference, integration of peaks in the chromatogram provides a total absorbance. Peak of interest (e.g. QS-21 main peak) is compared to total absorbance to determine peak content as a percentage.

The HPLC-UV method is also conveniently used to determine QS-21 main peak content and Preceding peak to QS-21 main peak ratio.

UPLC-UV Equipment

-   -   Waters Acquity UPLC     -   Waters Acquity Tunable UV Detector     -   Waters Acquity BEH C18 2.1×100 mm 1.7 um column     -   Mobile Phase A (MPA)—0.025% acetic acid in water/acetonitrile         (70:30 v/v)     -   Mobile Phase B (MPB)—0.025% trifluoroacetic acid in         water/acetonitrile (30:70 v/v)

TABLE 2 UPLC-UV Linear gradient conditions Time Flow rate (ml/min) % MPA % MPB 0 0.5 88 12 10.2 0.5 65.7 34.3 11.2 0.5 10 90 13.2 0.5 10 90

Column temperature 28 degrees C. 10 ul of sample is injected. UV detection is set at 214 nM.

Using a blank injection for reference, integration of peaks in the chromatogram provides a total absorbance. Peak of interest (e.g. QS-21 main peak) is compared to total absorbance to determine peak content as a percentage.

The UPLC-UV method is also conveniently used to determine 2018/QS-21 Ratio.

UPLC-UV/MS Equipment

-   -   Waters Acquity UPLC     -   Waters Acquity Tunable UV Detector     -   Waters Single-Quadrupole Mass Detector SQD1 (scanning range 1400         to 2040 M/Z)     -   Waters Acquity BEH C18 2.1×100 mm 1.7 um column     -   Mobile Phase A (MPA)—0.025% trifluoroacetic acid in         water/acetonitrile/isopropyl alcohol (75:20:5 v/v)     -   Mobile Phase B (MPB)—0.025% trifluoroacetic acid in         water/acetonitrile/isopropyl alcohol (10:72:18 v/v)

TABLE 3 UPLC-UV/MS Linear gradient conditions Time Flow rate (ml/min) % MPA % MPB 0 0.6 100 0 6.23 0.6 23 77

Test sample is prepared in 0.2% acetic acid in water/acetonitrile (70:30 v/v). Column temperature 55 degrees C. 10 ul of sample is injected. UV detection is set at 214 nM.

The term ‘QS-21 group’ is meant the triterpenoid glycosides identified from the B-isomer to the peak preceding the Iyo impurity in the UPLC-UV/MS methods described herein. Although retention times vary slightly between runs, the QS-21 group is located at approximately 3.8 min (QS-21 B-isomer) to approximately 4.5 minutes (prior to Iyo impurity peak, containing desarabinofuranosyl-QS-21 1856 A component).

Using a blank injection for reference, integration of peaks in the chromatogram that elute after the solvent front between 0.5 and around 5.50 minutes and do not appear in the blank is undertaken.

The monoisotope of the most abundant species is identified by combining TIC over the entire chromatogram to create a combined spectrum.

Ratio of QS-21 2002 A component to QS-21 1988 A component is calculated by comparing the ion current associated with the QS-21 2002 A component with the ion current associated with the QS-21 1988 A component within the QS-21 main peak.

FIG. 5 provides a chromatogram of an exemplary saponin extract. FIG. 6 shows expanded detail of the region including the QS-21 group and impurity peak.

FIGS. 7A and 7B provide extracted mass chromatograms for QS-21 1988 A (FIG. 7A) and QS-21 2002 A (FIG. 7B) molecular weight ions of an exemplary purified Quillaja saponaria saponin extract.

Example 3—Purification of a Crude Aqueous Extract of Quillaja saponaria

Crude aqueous extract of Quillaja saponaria material having a 2018 component to QS-21 main peak ratio of 0.064 or lower and a Preceding peak to QS-21 main peak ratio of 0.4 or lower, was treated with PVPP (1 kg PVPP per litre of crude aqueous extract). After adsorption the mixture was filtered to separate the PVPP and bound impurities from the liquor.

FIG. 2 provides an example HPLC-UV chromatogram for crude aqueous extract of Quillaja saponaria (used for Preceding peak to QS-21 main peak ratio determination and QS-21 main peak content).

FIG. 3 provides an example UPLC-UV chromatogram for crude aqueous extract of Quillaja saponaria (used for 2018 component to QS-21 main peak ratio determination).

Filtered liquor was concentrated and further purified by ultrafiltration/diafiltration using water and a 30 kD Hellicon membrane.

Resulting permeate was purified by reverse phase chromatography using a polystyrene resin (Amberchrom XT20).

TABLE 4 Reverse phase chromatography polystyrene resin gradient conditions Initial Final Duration % Eluent % Eluent % Eluent % Eluent Step (min) A B A B Injection + Rinse 11.3 100%  0% 100%  0% Gradient Elution 1 3.0 100%  0%  71%  29% Gradient Elution 2 50.0  71%  29%  53%  47% Gradient Elution 3 3.0  53%  47%  0% 100% Regeneration 10  0% 100%  0% 100% Gradient 3.0  0% 100% 100%  0% Equilibration 13.0 100%  0% 100%  0%

-   -   Eluent A: 5% Acetonitrile and 0.25% acetic acid     -   Eluent B: 90% Acetonitrile and 0.25% acetic acid     -   Column: 30 cm ID, approximately 17.7 to 20.5 L volume     -   Loading: 50-110 g per injection

Fractions were pooled to provide polystyrene purified saponin extract with a composition:

% QS-21 main peak≥18% (by HPLC)

and

2018 component/QS-21 main peak ratio≤0.054 (by UPLC-UV).

FIG. 4 provides an example UPLC-UV chromatogram for a polystyrene purified saponin extract pool.

The combined polystyrene purified fraction pool was further purified by reverse phase chromatography using a phenyl resin (EPDM).

TABLE 5 Reverse phase chromatography phenyl resin gradient conditions Duration Step (min) % Eluent C % Eluent B Injection + Rinse 2.0 100% 0% Isocratic Elution 58.0 100% 0% Regeneration 5.0  0% 100%  Equilibration 10.0 100% 0%

-   -   Eluent B: 90% Acetonitrile and 0.25% acetic acid     -   Eluent C: 35.2% acetonitrile and 0.25% acetic acid     -   Column: 45 cm ID, approximately 39.8 to 42.9 L volume     -   Loading: 13-21 g per injection

QS-21 containing fractions were pooled to provide phenyl purified saponin extract with a composition:

% QS-21 group≥98.5%

QS-21 main peak≥94.5%

2018 component≤2.7%

Main peak outside of the QS-21 group≤1% (by UPLC-UV/MS).

The combined phenyl purified saponin extract was concentrated by capture and release with reverse phase chromatography using a C8 resin (Lichroprep RP8) and the following conditions:

-   -   Loaded to column conditioned at 24% acetonitrile and 0.20%         acetic acid.     -   Eluted with 60% acetonitrile and 0.20% acetic acid.     -   11 cm column, approximately 0.87 to 0.97 L volume     -   Load: 50-142 g per injection

The C8 concentrated saponin extract was subjected to solvent exchange using ultrafiltration/diafiltration and a Pellicon 1 kDa membrane to reduce acetonitrile content below 21%.

The resulting solvent exchanged saponin extract was then lyophilised in a single step to provide a final purified saponin extract product.

The use of the process as described in Example 3 can consistently provide a purified saponin extract of Quillaja saponaria having a defined content in terms of QS-21 main peak and 2018 component, presenting a chromatographic profile comparable to the chromatograms shown in FIGS. 5 to 9 .

Example 4—Screening of Glucosidases for Deglucosylation of QS-18 to QS-21 Method Enzyme Selection

The enzyme family of hydrolases (E.C.3.2.1.-) that act on glycosidic bonds (‘glycoside hydrolases’ (GH) or ‘glycosidases’), contains at present approximately one million members having wide ranging activities across molecules containing glycans and polysaccharides. A typical QS-18 family molecule contains a number of such glycosidic bonds, with the presence of the 1-3 bond between the alpha-L-rhamnose on the linear tetrasaccharide and the branched terminal beta-D-glucose differentiating the QS-18 family from the QS-21 family. The specific hydrolysis of this bond by a beta-glucosidase, i.e. an enzyme with exo-beta-1,3-glucosidase activity (E.C. 3.2.1.21 and E.C. 3.2.1.58) will therefore convert QS-18 family components to QS-21 family components. Specific members of the glycoside hydrolase family having exo-beta-1,3-glucosidase activity were initially identified using the CAZy (Carbohydrate Active enZyme) database (www.cazy.com), with GH families 1, 3 and 5 purported to have enzyme members with the desired exo-beta-1,3 activity. All sequences annotated by CAZy from GH families 1,3 and 5 were obtained, and separate curated hidden Markov model profiles constructed for each which were then used to identify additional familial enzymes by searching the 209 million protein member Uniprot (www.uniprot.org) knowledgebase with the software HMMER (Eddy, 1998). In total, 22,594 sequences: 12,049, 9,278 and 1,267 representatives from GH families 1,3 and 5, respectively, were identified using this method. MMSeqs2 (Hauser, 2016) was then used to cluster each group of enzyme sequences using the default clustering workflow and parameters with a minimum sequence identity and coverage of 30% and 80%, respectively. In cases where the initial clustering yielded clusters with more than 1000 members, a second sub-clustering was performed at a higher 50% or 70% identity to ensure diverse exemplars from these larger clusters were represented more prominently. All clusters were then examined, and exemplars selected from each with preferences for annotation quality, known experimental activity, existing three dimensional structures from the Protein Data Bank (www.wwpdb.org) or known extremophile organisms as annotated by Uniprot. A final set of 400 diverse candidate enzymes was selected. Polynucleotide sequences encoding each selected enzyme linked to an N-terminal 6×His tag and Tev-cleavage site were prepared (amino acid sequence for His-tag linker, inserted N-terminally of normal start methionine, is provided in SEQ ID No. 1177) using a proprietary genetic-algorithm based codon optimization code.

Details of the candidate enzyme and polynucleotide sequences are summarised below in Table 6.

TABLE 6 Glucosidase candidate sequences AA DNA UniParc Ref @1 Uniprot ref Organism Seq ID Seq ID UPI00049B1A8C A0A061B3J2 Cyberlindnera fabianii  1 401 UPI0004E3EF7B A0A085EII0 Flavobacterium gilvum  2 402 UPI00050EE490 A0A090X649 Algibacter lectus  3 403 UPI0005ECB51E A0A0F0LB94 Microbacterium azadirachtae  4 404 UPI0006588DAD A0A0J0UT37 Actinobacteria bacterium  5 405 UPI0007968552 A0A136KWB3 Chloroflexi bacterium  6 406 UPI0002080410 A0A181C809 Komagataeibacter rhaeticus  7 407 UPI0008211BFC A0A1C5WEL8 Bacteroides sp.  8 408 UPI00085A2BD0 A0A1D8FZW3 Streptomyces rubrolavendulae  9 409 UPI00098C60F6 A0A1S8KYM5 Clostridium roseum  10 410 UPI0009CE0D4C A0A1V5M6V6 uncultured bacterium  11 411 UPI0009D4127D A0A1V6AN95 Firmicutes bacterium  12 412 UPI00045601AB A0A061H1Z3 Anthracocystis flocculosa  13 413 UPI0004FF77C7 A0A086ZKU2 Bifidobacterium boum  14 414 UPI00051EDBDE A0A098LTR2 Jejuia pallidilutea  15 415 UPI00062105AB A0A0F8B2B0 Ceratocystis fimbriata  16 416 UPI0006583AB1 A0A0J0UVW7 Actinobacteria bacterium  17 417 UPI0007AABFAD A0A143QAX3 Rhodococcus sp.  18 418 UPI0007F2D02D A0A194VF47 Valsa mali  19 419 UPI000821004D A0A1C5WSI4 uncultured Bacteroides sp.  20 420 UPI00084089B2 A0A1E3ALT2 Eisenbergiella tayi  21 421 UPI000978E914 A0A1V2MY14 Streptomyces sp.  22 422 UPI0009CBF21C A0A1V5MH90 Firmicutes bacterium  23 423 UPI0009D5B1F0 A0A1V6BAK3 Tenericutes bacterium  24 424 UPI0004A87350 A0A067Z479 Gluconobacter oxydans  25 425 UPI00050787A2 A0A087B8Q8 Bifidobacterium catenulatum  26 426 UPI0005965863 A0A0B7K538 Bionectria ochroleuca  27 427 UPI0006377CA4 A0A0G0GD78 Parcubacteria sp.  28 428 UPI0006228575 A0A0M2H276 Microbacterium ketosireducens  29 429 UPI0006C454B4 A0A173R3W4 Roseburia faecis  30 430 UPI0007F1D695 A0A1A6A050 Kwoniella dejecticola  31 431 UPI0008232A70 A0A1C6C862 uncultured Clostridium sp.  32 432 UPI000866CB30 A0A1E4S2F8 Cyberlindnera jadinii  33 433 UPI0009D3483D A0A1V5G4W6 Bacteroidetes bacterium  34 434 UPI0009CC9AEA A0A1V5Q4R4 Verrucomicrobia bacterium  35 435 UPI0009C6DE73 A0A1V6BV25 Bacteroidetes bacterium  36 436 UPI0004E051A9 A0A077WUK7 Lichtheimia ramosa  37 437 UPI0005060F52 A0A087BWT7 Bifidobacterium mongoliense  38 438 UPI000591CED9 A0A0B8NZY1 Vibrio ishigakensis  39 439 UPI00063B706F A0A0G2HEV5 Phaeomoniella chlamydospora  40 440 UPI0006C04F59 A0A0M8K5H7 Ardenticatena maritima  41 441 UPI000197E031 A0A173WPC4 Coprococcus comes  42 442 UPI0007DDBAB3 A0A1A9GNJ0 Nocardioides dokdonensis  43 443 UPI000822F7EB A0A1C6EBC1 uncultured Clostridium sp.  44 444 UPI000878EB40 A0A1F2PFB4 Acetobacterium wieringae  45 445 UPI0009D0A2C1 A0A1V5HNJ3 Tenericutes bacterium  46 446 UPI0009CB8A73 A0A1V5UFI1 Tenericutes bacterium  47 447 UPI0009CC90AF A0A1V6CDT2 uncultured bacterium  48 448 UPI0004D8E473 A0A078SYD0 Parabacteroides distasonis  49 449 UPI0005006C6A A0A087CJB0 Bifidobacterium psychraerophilum  50 450 UPI00059A3BA8 A0A0C2YJF2 Hebeloma cylindrosporum  51 451 UPI0006579FEA A0A0G4JRR8 Brenneria goodwinii  52 452 UPI00073C9E60 A0A0U5GN20 Aspergillus calidoustus  53 453 UPI0006C6E0C3 A0A174BEZ2 Bacteroides finegoldii  54 454 UPI0008153D6D A0A1B2A943 Altererythrobacter dongtanensis  55 455 UPI0008206E7B A0A1C6FWD9 uncultured Anaerotruncus sp.  56 456 UPI0008AC0E17 A0A1F5UJR6 Candidatus firestone  57 457 UPI0009C6C8E4 A0A1V5HS22 Tenericutes bacterium  58 458 UPI0009CEE1AB A0A1V5VBL2 Lentisphaerae bacterium  59 459 UPI0009CAAB0A A0A1V6FZ47 Planctomycetes bacterium  60 460 UPI0004DD62AC A0A084G332 Pseudallescheria apiosperma  61 46 UPI000507F00A A0A090Q4N8 Nonlabens sediminis  62 462 UPI0005CC42CA A0A0C5VDU3 Gynuella sunshinyii  63 463 UPI00063E4005 A0A0G4N9Q7 Verticillium longisporum  64 464 UPI00073C6CD3 A0A0V8TAB5 Cellulomonas sp.  65 465 UPI0006C024BB A0A174FBX7 Hungatella hathewayi  66 466 UPI000688E74D A0A1C2DG64 Mesorhizobium sp.  67 467 UPI000822FAB7 A0A1C6GRT5 Clostridium sp.  68 468 UPI0009284E74 A0A1M3CSY6 Chlamydiales bacterium  69 469 UPI0009CB461D A0A1V5HUX3 Spirochaetes bacterium  70 470 UPI0009C92D6A A0A1V5VHB9 bacterium  71 471 UPI0009D12B86 A0A1V6H2W5 Thermotogae bacterium  72 472 UPI0004DCBED3 A0A084GGE2 Pseudallescheria apiosperma  73 473 UPI00050E0BE3 A0A090VF17 Algibacter lectus  74 474 UPI0005B075C8 A0A0C9TWP5 Paxillus involutus  75 475 UPI00063DF006 A0A0G4NA55 Verticillium longisporum  76 476 UPI00076F8EA4 A0A110B1H1 Mucilaginibacter gotjawali  77 477 UPI0006C0FFEF A0A174IWW4 Bacteroides uniformis  78 478 UPI0008222B77 A0A1C5W0N6 Coprococcus sp.  79 479 UPI0006C36823 A0A1C6K2X5 Blautia sp.  80 480 UPI00092B3CD4 A0A1M3ELH4 Cellulomonas sp.  81 481 UPI0009C85428 A0A1V5J984 bacterium  82 482 UPI0009CA1D5C A0A1V5WHQ7 Spirochaetes bacterium  83 483 UPI0009C7041F A0A1V6IJ57 Tenericutes bacterium  84 484 UPI0004E2A41F A0A085EG29 Flavobacterium gilvum  85 485 UPI0005102470 A0A090WWZ1 Algibacter lectus  86 486 UPI0005B0F0AE A0A0C9WDY0 Hydnomerulius pinastri  87 487 UPI00065C2666 A0A0H5NWN2 Nocardia farcinica  88 488 UPI000760375B A0A125MG18 Bacteroides cellulosilyticus  89 489 UPI0007DF4250 A0A178ZBN2 Fonsecaea erecta  90 490 UPI0008209852 A0A1C5W9N9 Bacteroides sp.  91 491 UPI00086C34C1 A0A1D3UGH8 Tannerella forsythia  92 492 UPI00097E83BB A0A1R4KI92 Microbacterium esteraromaticum  93 493 UPI0009D255E5 A0A1V5LJK9 bacterium  94 494 UPI0009C5A3CF A0A1V5Z2L2 Candidatus hydrogenedentes  95 495 UPI0009C55799 A0A1V6J4J8 Bacteroidetes bacterium  96 496 UPI0009D4067F A0A1V6RQ41 Penicillium solitum  97 497 UPI0008737AA2 A0A288Q812 Weissella soli  98 498 UPI000CAC57D4 A0A2K4ZN91 Acetatifactor muris  99 499 UPI000D237A4A A0A2T2NYD4 Corynespora cassiicola 100 500 UPI000D77C91D A0A316V6M3 Meira miltonrushii 101 501 UPI00004E1F76 A0A380YVC7 Bacteroides fragilis 102 502 UPI000F0C30E8 A0A3G2SB79 Malassezia restricta 103 503 UPI000FFFEFB2 A0A430LYA2 Fusarium euwallaceae 104 504 UPI0010251887 A0A4Q2E070 Psathyrella aberdarensis 105 505 UPI0000E69509 A0KLP6 Aeromonas hydrophila 106 506 UPI00000B86CB A4F7P9 Saccharopolyspora erythraea 107 507 UPI000180240E B5I181 Streptomyces sviceus 108 508 UPI000A250F78 A0A1Y2AWB7 Naematelia encephala 109 509 UPI000C0221F1 A0A2C9D612 Hartmannibacter diazotrophicus 110 510 UPI000CEB5AB1 A0A2L2BPE2 Pontimonas salivibrio 111 511 UPI000D5B9C38 A0A2V1CH24 Cadophora sp. 112 512 UPI000D779558 A0A316V8S0 Meira miltonrushii 113 513 UPI000DC42E3D A0A395IJW4 Monilinia fructigena 114 514 UPI000F3E476C A0A3M6XGS0 Hortaea werneckii 115 515 UPI00101460D7 A0A445N7U0 Streptomyces netropsis 116 516 UPI001139C6A8 A0A4S9IFI0 Aureobasidium pullulans 117 517 UPI0000EA5CFF A1CTN9 Aspergillus clavatus 118 518 UPI0001523037 A5CT94 Clavibacter michiganensis 119 519 UPI0001831CF5 B6H7R5 Penicillium rubens 120 520 UPI000B365547 A0A1Y4NTL9 Lachnoclostridium sp. 121 521 UPI000C09BF88 A0A2D5IXB9 Rhodobacteraceae bacterium 122 522 UPI0004B5EEF2 A0A2M9UUC4 Bacteroides fragilis 123 523 UPI000D7FE1ED A0A2V5IY78 Aspergillus indologenus 124 524 UPI000D802B25 A0A316YR39 Acaromyces ingoldii 125 525 UPI000DC60823 A0A395J1U5 Monilinia fructigena 126 526 UPI000F52D773 A0A3N6CA02 Paenibacillus xylanexedens 127 527 UPI000F6DAAAE A0A448HIG0 Actinomyces howellii 128 528 UPI00113D19DF A0A4V5N914 Friedmanniomyces endolithicus 129 529 UPI0000EA8672 A1DNS0 Neosartorya fischeri 130 530 UPI0000D7314B A6V4K6 Pseudomonas aeruginosa 131 531 UPI00018E7266 B8MF24 Talaromyces stipitatus 132 532 UPI000A1A5FD7 A0A1Y5RVF8 Aquimixticola soesokkakensis 133 533 UPI000C98D37C A0A2D9YGV1 Rhodobacterales bacterium 134 534 UPI0006F9793E A0A2N9AS40 Methylorubrum extorquens 135 535 UPI00000CF7C8 A0A2X2YBP0 Clostridium perfringens 136 536 UPI0008AEBEA3 A0A348NID6 Acholeplasmatales bacterium 137 537 UPI000EC3C979 A0A3B8VHE8 Bacteroidetes bacterium 138 538 UPI000F637E38 A0A3P6K8E8 Clostridium carnis 139 539 UPI000B93B5C9 A0A448HNB5 Mycolicibacterium flavescens 140 540 UPI000F6EFA71 A0A4V6LJ94 Streptococcus gallolyticus 141 541 UPI0000EB54CC A1JNB7 Yersinia enterocolitica 142 542 UPI0000ED8A80 A7HFG4 Anaeromyxobacter sp. 143 543 UPI00018E7D70 B8MK55 Talaromyces stipitatus 144 544 UPI000A2E3FAA A0A1Z5SL14 Hortaea werneckii 145 545 UPI000C529025 A0A2E2Q8X2 Micavibrio sp. 146 546 UPI000CE6B996 A0A2N9MBS0 Acidobacteriia bacterium 147 547 UPI000DA3A972 A0A2X4T570 Corynebacterium jeikeium 148 548 UPI000E8D37A1 A0A353PZH8 Clostridiaceae bacterium 149 549 UPI000748C096 A0A3B9PA35 Anaerolineaceae bacterium 150 550 UPI000FF41956 A0A409WSY0 Gymnopilus dilepis 151 551 UPI000F7105D4 A0A448R8N0 Kocuria rosea 152 552 UPI0011696FAB A0A509DWZ3 Teredinibacter sp. 153 553 UPI0000EFB564 A2QS42 Aspergillus niger 154 554 UPI000164423D B0D734 Laccaria bicolor 155 555 UPI00017357F6 B9XH33 Pedosphaera parvula 156 556 UPI00042469F6 A0A239TGH2 Megamonas hypermegale 157 557 UPI000BC209C3 A0A2H3E300 Armillaria gallica 158 558 UPI000D2EAE87 A0A2P8AV03 Micromonospora sp. 159 559 UPI000DA286EE A0A2X5CJC5 Klebsiella oxytoca 160 560 UPI000DFAEF6C A0A367ZIH8 Candidatus ozemobacter 161 561 UPI000E399EE8 A0A3D8R2C2 Coleophoma crateriformis 162 562 UPI000FBC01E3 A0A427XHS2 Apiotrichum porosum 163 563 UPI00068E4E50 A0A449BJ27 Acholeplasma hippikon 164 564 UPI001185F074 A0A516RGT1 Streptomyces spectabilis 165 565 UPI0000EFCED2 A2R8G2 Aspergillus niger 166 566 UPI00000D8BFA B0RYA0 Xanthomonas campestris 167 567 UPI00019C9CD7 C2FDL2 Lactobacillus paracasei 168 568 UPI000BC490A3 A0A261XUH4 Bifiguratus adelaidae 169 569 UPI000CC31AE2 A0A2H5YYA1 bacterium 170 570 UPI000D26C9C6 A0A2P9IY35 Actinomadura parvosata 171 571 UPI00024F22B8 A0A2Z5Y4P3 Melissococcus plutonius 172 572 UPI000E020871 A0A377KJS3 Enterococcus durans 173 573 UPI000DD17A7D A0A3G2S2J6 Malassezia restricta 174 574 UPI000FA2DE87 A0A427XZQ0 Apiotrichum porosum 175 575 UPI0010355193 A0A4P6PWP5 Streptomonospora sp. 176 576 UPI00119640A1 A0A558LH47 Lactobacillus gasseri 177 577 UPI0000EFD0AA A2RAJ1 Aspergillus niger 178 578 UPI000170BF91 B0XXG1 Neosartorya fumigata 179 579 UPI0001A5B234 C4Z6T5 Eubacterium eligens 180 580 UPI000BC64A75 A0A261XVM8 Bifiguratus adelaidae 181 581 UPI000CAA854E A0A2H6EX57 bacterium 182 582 UPI000D24EC2C A0A2T2N5H9 Corynespora cassiicola 183 583 UPI000D77A0B7 A0A316UK83 Pseudomicrostroma glucosiphilum 184 584 UPI0002DAAFCC A0A380HD31 Staphylococcus saprophyticus 185 585 UPI000F0C6B8E A0A3G2S932 Malassezia restricta 186 586 UPI000FBA70E2 A0A427YCL1 Saitozyma podzolica 187 587 UPI00102822B4 A0A4Q1BRC6 Tremella mesenterica 188 588 UPI0000527506 A0JZ86 Arthrobacter sp. 189 589 UPI000157388C A3LRB0 Scheffersomyces stipitis 190 590 UPI0001712E02 B1XZK8 Leptothrix cholodnii 191 591 UPI000166883C C4ZLL7 Thauera sp. 192 592 UPI00018494AB C5CDW5 Kosmotoga olearia 193 593 UPI0001CD671F D4L3Y2 Roseburia intestinalis 194 594 UPI0001E0DC00 E0PDF8 Streptococcus equinus 195 595 UPI0001F80C4B E8JUK5 Streptococcus cristatus 196 596 UPI0001D2DBBC F2JLH3 Cellulosilyticum lentocellum 197 597 UPI0001C48657 F5WY15 Streptococcus gallolyticus 198 598 UPI00021D443A F9Y8Z7 Ketogulonicigenium vulgare 199 599 UPI000228255D G3AGX1 Spathaspora passalidarum 200 600 UPI00023F6F5F G8T9J3 Niastella koreensis 201 601 UPI000260108C I3IDC0 Cellvibrio sp. 202 602 UPI000272D1E0 J1ACAb Flavobacterium sp. 203 603 UPI00028E7FE1 K2S5D3 Macrophomina phaseolina 204 604 UPI00018483A2 C5CHI5 Kosmotoga olearia 205 605 UPI0001CD5918 D4LRF6 Blautia obeum 206 606 UPI0001E18CDA E0Q541 Bifidobacterium dentium 207 607 UPI0001F55F8F E8N5R8 Anaerolinea thermophila 208 608 UPI0002050DA2 F2N7E4 Coriobacterium glomerans 209 609 UPI000210C886 F5XJQ3 Microlunatus phosphovorus 210 610 UPI0001C18877 G0Q1U8 Streptomyces sp. 211 611 UPI0002282B61 G3AIV6 Spathaspora passalidarum 212 612 UPI0002402F4A H0ET34 Glarea lozoyensis 213 613 UPI0002633B36 I3UJK0 Glaciozyma antarctica 214 614 UPI00027DFD35 J7LN00 Arthrobacter sp. 215 615 UPI00028D698C K3VMA9 Fusarium pseudograminearum 216 616 UPI000006AA61 C6GGC9 Hypocrea rufa 217 617 UPI0001CDA964 D4M6W6 Ruminococcus torques 218 618 UPI0001E17370 E0Q9Z7 Bifidobacterium dentium 219 619 UPI0001FC71F6 F0R2D7 Bacteroides salanitronis 220 620 UPI00020E6D19 F3ZQ40 Bacteroides coprosuis 221 621 UPI00020D4CC6 F6CWF6 Marinomonas posidonica 222 622 UPI000227E8ED G0SE64 Chaetomium thermophilum 223 623 UPI00022B9A15 G4L5K1 Tetragenococcus halophilus 224 624 UPI00024F0867 H6CCZ8 Paenibacillus sp. 225 625 UPI000265AA4B I4B8U7 Turneriella parva 226 626 UPI00027DFDB2 J7LQK9 Arthrobacter sp. 227 627 UPI00029074B1 K5W7V1 Agaricus bisporus 228 628 UPI0001B67634 C7YIP3 Nectria haematococca 229 629 UPI0001A25287 D4VSZ0 Bacteroides xylanisolvens 230 630 UPI0001DD9790 E1R331 Sediminispirochaeta smaragdinae 231 631 UPI0001FC42E9 F0RPV2 Deinococcus proteolyticus 232 632 UPI0002033A0A F4C226 Sphingobacterium sp. 233 633 UPI0001E54133 F6ET40 Sphingobium chlorophenolicum 234 634 UPI00021CACC4 G0V3V5 Caloramator australicus 235 635 UPI000230E3BF G6F370 Commensalibacter intestini 236 636 UPI00024F07AC H6CIT2 Paenibacillus sp. 237 637 UPI0002638AF3 I4EIA9 Nitrolancea hollandica 238 638 UPI000392C3ED J9VVK7 Cryptococcus neoformans 239 639 UPI0002988588 K7S596 Acidipropionibacterium acidipropionici 240 640 UPI0001B93465 C9PT75 Prevotella sp. 241 641 UPI0001D08095 D5ALU0 Rhodobacter capsulatus 242 642 UPI0001E74370 E3FJ05 Stigmatella aurantiaca 243 643 UPI0002010060 F0RVK3 Sphaerochaeta globosa 244 644 UPI000207D78C F4GH96 Sphaerochaeta coccoides 245 645 UPI00020EFBCB F6ICQ5 Novosphingobium sp. 246 646 UPI000225331F G1XH86 Arthrobotrys oligospora 247 647 UPI00000C6907 G6FFS4 Lactococcus lactis 248 648 UPI000255313A H8FXH7 Phaeospirillum molischianum 249 649 UPI0002609C68 I4EW72 Modestobacter marinus 250 650 UPI00028AC337 K0K125 Saccharothrix espanaensis 251 651 UPI00029BA293 K8DAK0 Cronobacter sakazakii 252 652 UPI0001BBDF1E C9SVX1 Verticillium alfalfae 253 653 UPI0001CCFD36 D6CY10 Bacteroides xylanisolvens 254 654 UPI0001EBD98A E4RUR0 Leadbetterella byssophila 255 655 UPI00020102A5 F0RYB6 Sphaerochaeta globosa 256 656 UPI000207D6A0 F4GLH6 Sphaerochaeta coccoides 257 657 UPI000212252C F7Q0Y2 Haloplasma contractile 258 658 UPI0002265447 G1Y4C7 Nitrospirillum amazonense 259 659 UPI00023420B8 G8AWD9 Azospirillum brasilense 260 660 UPI000255314F H8FXJ8 Phaeospirillum molischianum 261 661 UPI000260A2FA I4EYD5 Modestobacter marinus 262 662 UPI000283EB8D K0KVJ2 Wickerhamomyces ciferrii 263 663 UPI0002A5D085 K9XKL8 Gloeocapsa sp. 264 664 UPI0001A3BCB6 D1C7U8 Sphaerobacter thermophilus 265 665 UPI0001CCEF1F D6D4V2 Bacteroides xylanisolvens 266 666 UPI0001F149E8 E6K4W5 Prevotella buccae 267 667 UPI0001FF1101 F0XBR0 Grosmannia clavigera 268 668 UPI00020F9774 F4R4W2 Melampsora larici-populina 269 669 UPI0002138E9C F8N7G1 Prevotella multisaccharivorax 270 670 UPI0002255A63 G2G8K4 Streptomyces zinciresistens 271 671 UPI0001D9FC40 G8NY42 Granulicella mallensis 272 672 UPI00021F1FEE I1RH94 Gibberella zeae 273 673 UPI00026090A2 I4EYK6 Modestobacter marinus 274 674 UPI0002869F55 K0NRS8 Lactobacillus equicursoris 275 675 UPI0002A93280 L2F9W0 Colletotrichum fructicola 276 676 UPI0001BF8AF6 D2B261 Streptosporangium roseum 277 677 UPI00019F252A D7UX19 Listeria grayi 278 678 UPI0001F11EFF E6LF07 Enterococcus italicus 279 679 UPI000203D9EA F2IIT6 Fluviicola taffensis 280 680 UPI00020CA641 F5N4W9 Shigella flexneri 281 681 UPI0002189080 F9EFW5 Actinomyces sp. 282 682 UPI00022EBE5A G2X5V6 Verticillium dahliae 283 683 UPI00023ED5A8 G8S0M0 Actinoplanes sp. 284 684 UPI000023EA29 I1S320 Gibberella zeae 285 685 UPI00027CE685 J0WV06 Auricularia subglabra 286 686 UPI00028E86D6 K2N268 Nitratireductor indicus 287 687 UPI0002AD999E L7VH66 Thermoclostridium stercorarium 288 688 UPI0002AF2DE3 L8A1S2 Geobacillus sp. 289 689 UPI000327C3CC M9ZC55 uncultured bacterium 290 690 UPI000059ACE1 Q0BAK2 Burkholderia ambifaria 291 691 UPI0000676B8D Q2UIR4 Aspergillus oryzae 292 692 UPI00004269B5 Q66DJ0 Yersinia pseudotuberculosis 293 693 UPI000331CC98 R9AF64 Wallemia ichthyophaga 294 694 UPI0003522404 S3DIM6 Glarea lozoyensis 295 695 UPI0003BF753A V2YU31 Moniliophthora roreri 296 696 UPI0003DBCB4F W2ULL3 Zhouia amylolytica 297 697 UPI0003F362FF W9NQQ3 Fusarium oxysporum 298 698 UPI00006EA168 A5FAA5 Flavobacterium johnsoniae 299 699 UPI00002394F5 Q75193 Oryza sativa 300 700 UPI0002B65681 M1MCD1 Clostridium saccharoperbutylacetonicum 301 701 UPI0002D21DB1 N1MBN6 Rhodococcus sp. 302 702 UPI000059C0CD Q0BCV8 Burkholderia ambifaria 303 703 UPI00005CE9E7 Q3BVH7 Xanthomonas campestris 304 704 UPI00000C7604 Q9A6F8 Caulobacter vibrioides 305 705 UPI000338772A R9GRA7 Arcticibacter svalbardensis 306 706 UPI00035A4604 S7VQ28 Winogradskyella psychrotolerans 307 707 UPI0003C4ED6A V5DXT8 Methyloglobulus morosus 308 708 UPI0003E05C04 W3WWV37 Pestalotiopsis fici 309 709 UPI000434E698 W9YNR7 Capronia coronata 310 710 UPI00001268FD P48825 Aspergillus aculeatus 311 711 UPI0000DD5996 Q0GC07 Thermotoga neapolitana 312 712 UPI0002B666E8 M1MJF4 Clostridium saccharoperbutylacetonicum 313 713 UPI000006B001 O74799 Schizosaccharomyces pombe 314 714 UPI000161BD2B Q0TXF6 Phaeosphaeria nodorum 315 715 UPI00003806C8 Q3RGJ3 Xylella fastidiosa Dixon 316 716 UPI000006A330 Q9P6J6 Schizosaccharomyces pombe 317 717 UPI000337E887 R9GWD6 Arcticibacter svalbardensis 318 718 UPI0003885717 T0KJI7 Colletotrichum gloeosporioides 319 719 UPI0003C9E340 V5R1E8 uncultured bacterium 320 720 UPI0003E06A61 W4S715 Xanthomonas arboricola 321 721 UPI00021EC697 X0A8X8 Fusarium oxysporum 322 722 UPI00001108D0 P22073 Paenibacillus polymyxa 323 723 UPI0001BE5ADA D1GCC6 Kluyveromyces marxianus 324 724 UPI0002C04A25 M5A594 Ilumatobacter coccineus 325 725 UPI0000126912 P12614 Agrobacterium sp. 326 726 UPI000038ECF7 Q11P53 Cytophaga hutchinsonii 327 727 UPI00003C5CA4 Q47PF5 Thermobifida fusca 328 728 UPI0000069E5E Q9UVJ6 Botryotinia fuckeliana 329 729 UPI00033994D2 R9PTK2 Agarivorans albus 330 730 UPI00038B7639 T0UDU2 Enterococcus sp. 331 731 UPI0003D8A7DC V5WKT4 Salinispira pacifica 332 732 UPI0003ECF15E W6P696 Bacteroides xylanisolvens 333 733 UPI000430029A X0AB46 Fusarium oxysporum 334 734 UPI0000126906 Q08638B Thermotoga maritima 335 735 UPI0000083EA8 Q8T0W7 Neotermes koshunensis 336 736 UPI0002BF34B3 M5CHG9 Thanatephorus cucumeris 337 737 UPI000053581D P14002 Hungateiclostridium thermocellum 338 738 UPI0000D76A70 Q1IJ89 Koribacter versatilis 339 739 UPI00005203D8 Q4WLX5 Neosartorya fumigata 340 740 UPI00032DB8C1 R1FKF0 Amycolatopsis vancoresmycina 341 741 UPI0003519C76 S0EKU1 Gibberella fujikuroi 342 742 UPI000390F839 T2I2H5 Bifidobacterium longum 343 743 UPI0003DFF0DA W0FLD1 uncultured bacterium 344 744 UPI0002D3D994 W6P9J9 Bacteroides xylanisolvens 345 745 UPI00042F8016 X0HA35 Fusarium oxysporum 346 746 UPI00000A7F40 Q41290 Sorghum bicolor 347 747 UPI000050B701 Q53EH2 Clostridium cellulovorans 348 748 UPI0002C29EAA M5G1U5 Dacryopinax primogenitus 349 749 UPI0000126913 P27034 Rhizobium radiobacter 350 750 UPI00006E0CC7 Q25BW5A Phanerochaete chrysosporium 351 751 UPI0000126905 Q59437 Enterobacter agglomerans 352 752 UPI00033056C4 R4YWU4 Candidatus microthrix 353 753 UPI0003427F29 S0ET37 Chthonomonas calidirosea 354 754 UPI0003B04208 U4KMR7 Acholeplasma brassicae 355 755 UPI0003E13BD3 W0LJ28 Chania multitudinisentens 356 756 UPI0003ECCF80 W7MW98 Gibberella moniliformis 357 757 UPI0004305856 X0I2U8 Fusarium oxysporum 358 758 UPI00006E0CC7 Q25BW5B Phanerochaete chrysosporium 359 759 UPI00000A7EFC Q9FYS3 Secale cereale 360 760 UPI0002C0273E M5QUM2 Anoxybacillus gonensis 361 761 UPI0000126906 Q08638A Thermotoga maritima 362 762 UPI00003C2ACC Q2RP51 Rhodospirillum rubrum 363 763 UPI00000B9013 Q60038 Thermotoga neapolitana 364 764 UPI00032F466E R4Z6M6 Candidatus microthrix 365 765 UPI00032874EC S0FPI8 Ruminiclostridium cellobioparum 366 766 UPI0003B0490C U4KMV4 Acholeplasma brassicae 367 767 UPI0003DB6EFD W1BBP5 Klebsiella pneumoniae 368 768 UPI00045BB507 X8JI88 Rhizoctonia solani 370 770 UPI0000072C73 Q9H227 Homo sapiens 371 771 UPI0000DD5304 Q0GMU3 uncultured bacterium 372 772 UPI0002CA9E4F M8CQD9 Thermoanaerobacter thermohydrosulfuricus 373 773 UPI0000E297D8 Q091M8 Stigmatella aurantiaca 374 774 UPI00006676B1 Q2T7C9 Burkholderia thailandensis 375 775 UPI0000426E8F Q665S2 Yersinia pseudotuberculosis 376 776 UPI00032BF71F R8BQM8 Togninia minima 377 777 UPI00035213C2 S3BVJ8 Ophiostoma piceae 378 778 UPI0003B117B7 U4LIL3 Pyronema omphalodes 379 779 UPI0001F76D1F W1QIW0 Ogataea parapolymorpha 380 780 UPI0003F2059F W8F6U0 Hymenobacter swuensis 381 781 UPI00006E8E1B A5FEF5 Flavobacterium johnsoniae 382 782 UPI000012690B P22505 Paenibacillus polymyxa 383 783 UPI000009D014 Q8L7J2 Oryza sativa 384 784 UPI0000E580F2 B8AVF0 Oryza sativa 385 785 UPI000EA7F16F A0A452CSM4 Nannochloris 386 786 UPI00006AE508 B8CYA8 Halothermothrix orenii 387 787 UPI000018B2B4 Q7RWP2 Neurospora crassa 388 788 UPI000192BB5F B9V8P5 Micrococcus antarcticus 389 789 UPI000285E79E K0A8J9 Exiguobacterium antarcticum 390 790 UPI00000BEB61 Q9RA61 Thermus thermophilus 391 791 UPI00078BF747 A0A2T4AR08 Trichoderma harzianum 392 792 UPI000006AA61 Q12715 Hypocrea jecorina 393 793 UPI00000B411B Q59976 Streptomyces sp. 394 794 UPI00000C7E56 Q99YP9 Streptococcus pyogenes 395 795 UPI000012691B P26205 Trifolium repens 396 796 UPI000006C8FF Q8TGI8 Talaromyces emersonii 397 797 UPI0000126903 P26208 Hungateiclostridium thermocellum 398 798 UPI000219FE3E F9ULH8 Lactobacillus plantarum 399 799 UPI0003F2033A A0A2I4PGZ0 Agrobacterium tumefaciens 400 800 Experiment 4-1—Screening of Glucosidases for Deglycosylation with Purified QS-18 (0.04 mg/ml) at pH 7.5 and Room Temperature

Nucleotide sequences were sub-cloned into pET24b+ for expression. 10 uL of E. coli cells (One Shot® BL21(DE3) chemically competent E. coli) were transferred to each well of a 96 well PCR plate (prechilled on ice). 10 uL autoclaved water was added to the DNA, resuspended by pipetting, and 1 uL of plasmid DNA (15-30 ng) was transferred to the competent cells. Immediately after addition, the resulting mixture was mixed by pipetting. Cells were heat shocked by placing the plate in a thermal cycler at 42° C. for 30 seconds then transferred directly to an ice bath for 2 min. 100 uL sterile Lysogeny Broth (LB) medium was added to each well containing transformed cells. The content of each plate was transferred into a 96-deep well plate pre-aliquoted with 400 uL LB and the plate was incubated at 37° C. with shaking and 85% humidity for 1 hour. After outgrowth, 500 uL of sterile LB containing 100 ug/mL kanamycin was added to the plates containing cells and plates incubated at 37° C. with shaking overnight (18 hours) with humidity control (85%).

1000 mLs of Overnight Express Media was supplemented with 1 mL kanamycin 50 mg/mL and 20 mL of 50% v/v glycerol added (50 ug/mL kanamycin final and 1% glycerol). 96-Well Assay Block 2 mL plates were aliquoted with 380 uL of media per well. Pre-inoculum (20 uL) from transformation plates was added. The plates were sealed appropriately and incubated at 37° C. at 300 with shaking for 2 h. After 2 h the temperature was lowered to 20° C. and incubation continued for 20 h.

The liquid cultures were centrifuged for 10 minutes at 4° C. The supernatant was discarded, plates blotted on an absorbent material to remove residue and the plates frozen at −80° C.

Lysis buffer was prepared according to the following protocol:

-   -   1. Polymyxin B sulphate (0.5 mg/mL) was suspended in 100 mM         potassium phosphate pH 7.5     -   2. The mixture was sonicated until polymyxin B sulphate had         dissolved     -   3. Lysozyme (1 mg/mL) and benzonase (0.1 uL/mL lysis buffer)         were added

2 copies of each cell pellet plate were removed from −80 degC freezer and allowed to thaw. 200 uL of lysis buffer was added to each well of one copy of the cell pellet plates. Plates were shaken at room temperature for 10 mins. 190 uL of cell pellet/lysis buffer was transferred to a corresponding fresh cell pellet plate. These plates were incubated at room temperature with shaking for 2 hours. Lysate was clarified by centrifugation (10 min, 4 degC).

QS-18 was obtained by analogous methods to Example 3, collecting a QS-18 containing phenyl fraction following phenyl treatment (presence of m/z corresponding to key components was confirmed by MS and the phenyl fraction then used without further treatment). QS-18 solution was prepared by diluting aqueous QS-18 (ca 1 mg/mL) with 100 mM potassium phosphate pH 7.5 to 0.2 mg/ml. 40 uL clarified lysate was transferred into fresh 96 well PCR plates. 10 uL QS-18 solution added to each well of lysate to a final concentration of 0.04 mg/ml. Incubated at room temperature with shaking for 20 h. Quenched with 50 uL MeCN and shaken at room temperature for 10 mins. Samples were analysed by LC-MS/MS using a Waters Acquity H class coupled to a Waters Xevo Tandem Quadrupole (TQD) Mass Spectrometer.

LCMS/MS 3 Min Method

UPLC Conditions Column Waters BEH C18 1.7 um 2.1 × 50 mm Temperature 40° C. ± 3° C. Flow Rate 0.5 mL/min Gradient Curve 6 Injection Volume 1 μL Autosampler Temperature 10° C. ± 3° C. Run Time 3 mins Post-Inject Wash 50% v/v methanol Mobile Phase A 0.1% v/v Formic Acid in Water Mobile Phase B 0.1% v/v Formic Acid in Acetonitrile Syringe Wash Solvent 50% v/v Methanol Seal Wash 50% v/v Methanol Purge Solvent 50% v/v Acetonitrile Gradient Time %A %B 0 55 45 0.75 55 45 2.00  5 95 2.1  5 95 2.2 55 45 3.0 55 45 Mass Spec Conditions (or similar to give maximum sensitivity) Source Analyser Extended Tab Capillary Voltage 3.9 kV LM Resolution 1 11.6 Source Temp 150° C. Cone 36 V HM Resolution 1 14.9 Extractor   3 V Desolvation 500° C. Ion Energy 1 0.4 RF Lens 2.5 V Temperature Source Temperature 150° C. LM Resolution 2 12.2 Entrance 30 Source Gas Flow HM Resolution 2 14.8 Exit 30 Desolvation Gas Flow 1000 L/Hr Ion Energy 2 0.8 Gain  1 Cone (L/Hr) 0 Collision Energy  3 Collision Gas Argon

Dwell Cone Collision Time Voltage Energy Component Mode (secs) MRM Transitions (V) (v) QS-18 2150 ESI - Auto 1074.50 > 836.4 34 16 QS-21 1988 ESI - Auto  993.46 > 755.5 34 16 Enzyme activity was calculated as:

${\%{conversion}} = \frac{100 \times {QS} - 21{peak}{area}}{\left( {{{QS} - 21{peak}{area}} + {{QS} - 18{peak}{area}}} \right)}$

The negative control reactions, which utilised a plasmid expressing an unrelated protein, had an average % conversion of 0.42% with a standard deviation (S.D.) of 0.10%. Candidate enzymes with % conversion >0.72%, i.e. >3 S.D. above negative control, were considered to be positive hits and are listed below in Table 7. Sample results are shown in FIG. 10 for a QS-21 standard, FIG. 11 for negative control and FIG. 12 for treatment with the glucosidase of SEQ ID No. 262.

Experiment 4-2—Screening of Glucosidases for Deglycosylation with Purified QS-18 (1 mg/ml) at pH 6 and 30 Deg C.

Lysates were prepared in an identical manner to Experiment 4-1 above, except the lysis buffer was prepared in 100 mM potassium phosphate buffer pH 6. QS-18 solution was prepared by dissolving QS-18 in 100 mM potassium phosphate buffer pH 6 (2 mg/mL).

12 uL clarified lysate was transferred into fresh 96 well PCR plates. 12 uL QS-18 solution was added (1 mg/ml final concentration), plates sealed and incubated overnight (30° C.) for 18 hrs. After quenching with 25 uL MeCN and shaking for 10 mins (RT), samples were analysed using the LC-MS/MS protocol described in Experiment 4-1 and enzyme activity determined in an analogous manner.

The negative control reactions had an average % conversion of 0.38% with a standard deviation (S.D.) of 0.06%. Sequences with % conversion >0.56% i.e. >3 S.D. above negative control are listed in Table 7.

Experiment 4-3—Screening of Glucosidases for Deglycosylation with QS-18 in Crude Bark Extract (1 in 2000 Dilution) at pH 7 and 30 Deg C.

Lysates were prepared according to the following procedure.

50 uL of 50% v/v glycerol was transferred to each well of a flat bottom 96 well plate. 50 uL from each well of the overnight culture plate (in LB) from Experiment 4-1 was transferred and mixed by pipette aspiration. The plate was then covered with a foil seal and frozen at −80° C. as a glycerol stock of the transformants. Glycerol stock plates were removed from −80° C. freezer and allowed to thaw. Overnight cultures were prepared by pipetting 5 mL LB into 50 mL tubes with Kanamycin as a selection marker at a final concentration of 50 μg/mL. Cultures were inoculated with 10 μL of glycerol stock and incubated overnight at 37° C. with shaking.

Flask cultures were prepared by pipetting 25 mL Terrific Broth (TB) into 250 mL conical flasks with Kanamycin as selection marker at a final concentration of 50 μg/mL. Overnight cultures OD₆₀₀ was measured using a spectrophotometer and initial inoculum volume calculated for a starting OD˜0.1. Cultures were inoculated and incubated at 37° C. with shaking up to OD˜0.6.

Cultures were induced with 1 mM IPTG and temperature was reduced to 20° C. with shaking. Cultures were then incubated overnight. Cultures were harvested in individual 1 mL aliquots (in 2 mL tubes). 1 mL aliquots were centrifuged at 13000 g for 3 min and supernatant discarded. Pellets were frozen at −20° C.

Lysis buffer was prepared according to the following protocol:

-   -   Polymyxin B sulphate (0.5 mg/mL) and Lysozyme (1 mg/mL) were         suspended in 50 mM phosphate buffer, 0.3 M NaCl, pH 8.     -   Benzonase (20 U/mL) and 0.05% Tween-20 were added.

1 mL of Lysis buffer was added to a pellet from 1 mL culture aliquot. Lysed samples were incubated at room temperature with shaking for 2 hours. Lysate was clarified by centrifugation at 13000 g, 5 min, 4° C.

Crude bark extract (CBE) obtained by aqueous extraction of Quillaja saponaria and containing at least 2.80 mg/ml QS-21 (by HPLC-UV) was diluted 1 in 400 in 50 mM potassium phosphate buffer at pH 7. 100 ul diluted CBE was added to 400 ul of each lysate to give a final dilution of 1 in 2000.

As diluted CBE was added to the lysate, the solution was vortexed for ˜5 seconds and then a 80 ul sample taken and quenched with 160 ul methanol (MeOH). This was used as a time 0 sample. The reaction solutions and controls were then left to shake at 30° C. Samples were taken after 1 h in the same way as the time 0 sample. Samples were analysed by LCMS/MS using the protocol described in Experiment 4-1 and enzyme activity determined in an analogous manner.

Enzyme activity is calculated as the % conversion of the QS-18 present in the crude bark extract:

${\%{conversion}} = \frac{100 \times \left( {{\%{QS} - 18_{t = 0}} - {\%{QS} - 18_{t = {1{hr}}}}} \right)}{\%{QS} - 18_{t = 0}}$ where ${\%{QS} - 18} = \frac{100 \times {QS} - 18{peak}{area}}{\left( {{{QS} - 21{peak}{area}} + {{QS} - 18{peak}{area}}} \right)}$

Experiment 4-4—Screening of Glucosidases for Deglycosylation of QS-18 in Crude Bark Extract (1 in 20 Dilution) to QS-21

Lysates were prepared as in Experiment 4-2.

Crude bark extract (CBE) was adjusted to pH 6 by dropwise addition of 2M NaOH with stirring. 25 uL of clarified lysate was transferred to a reaction plate, 22.5 ul of 100 mM potassium phosphate buffer pH 6 was added, 2.5 ul CBE at pH 6 was added. Reaction plates were sealed, incubated at 25 degC with shaking for 18 hours, then quenched by addition of 50 uL acetonitrile (MeCN). Quenched reaction plates were re-sealed and incubated at 20 degC with shaking for 10 min. The reaction plates were centrifuged (10 min, 4 degC) and analysed by UV HPLC with the method below:

UV Method EM2020N435545v1_2

Column details ACQUITY UPLC BEH C18 Column, 130 Å, 1.7 μm, 2.1 mm × 50 mm Column temperature 40° C. Mobile phase A 0.05% v/v TFA in Water HPLC Mobile phase B 0.05% v/v TFA in Acetonitrile HPLC Flow rate 1 mL per minute Time % Mobile % Mobile (mins) Phase A Phase B Gradient profile 0.0 97 3 5.5 min gradient 3.7 5 95 4.0 5 95 4.1 97 3 5.5 97 3 Detector type UV detector: DAD or VWD Detector wavelength 214 nm Agilent DAD 1260 4.0 nm (default setting) and 1290 Bandwidth Agilent 1290 and 1260 Off DAD reference wavelength Injection volume Default injection volume used is 1 ul

Three key peaks of interest are apparent using this chromatography: Left Peak (retention time approximately 2.30-2.35 min) comprising mainly QS-17 family components; Middle Peak (retention time approximately 2.37-2.42 min) comprising mainly QS-18 family components and desglucosyl-QS-17 family components; and Right Peak (retention time approximately 2.44-2.50 min) comprising mainly QS-21 family components. Peak identity was supported by MS/MS.

Enzyme activity is calculated as the % conversion of the Middle Peak present in the crude bark extract:

${\%{conversion}} = \frac{100 \times \left( {{\%{Middle}{Peak}_{t = 0}} - {\%{Middle}{Peak}_{t = {1{hr}}}}} \right)}{\%{Middle}{Peak}_{t = 0}}$ where ${\%{Middle}} = \frac{100 \times {Middle}{Peak}{area}}{\left( {{{Right}{Peak}{area}} + {{Middle}{Peak}{area}}} \right)}$

FIG. 13 provides exemplary chromatograms following glucosidase SEQ ID No. 262 treatment and for the negative control.

Results

TABLE 7 Expt 4-1 Expt 4-2 Expt 4-3 Expt 4-4 AA Seq ID DNA Seq ID % conv % conv % conv % conv 262 662 99.5 97.9 99.7 68.6 208 608 100.0 94.0 26.0 <3 63 463 95.6 54.2 N.T. <3 229 629 91.1 14.2 N.T. <3 250 650 88.5 36.0 2.6 <3 5 405 70.8 16.6 1.4 <3 101 501 28.2 1.0 1.8 <3 207 607 27.4 4.4 0.2 <3 169 569 19.8 2.1 2.4 <3 247 647 18.0 2.0 2.7 <3 302 702 17.2 2.1 N.T. <3 324 724 13.6 0.9 N.T. <3 319 719 10.2 0.4 N.T. <3 9 409 9.2 0.9 N.T. <3 240 640 9.0 0.5 N.T. <3 325 725 6.3 0.9 N.T. <3 338 738 6.3 0.5 N.T. <3 116 516 4.7 0.5 N.T. <3 41 441 4.0 0.7 N.T. N.T. 133 533 3.7 0.4 N.T. <3 350 750 2.6 0.7 N.T. <3 234 634 2.5 0.7 N.T. <3 380 780 2.4 0.5 N.T. <3 32 432 2.2 0.6 N.T. N.T. 312 712 1.6 0.4 N.T. N.T. 222 622 1.6 0.4 N.T. <3 354 754 1.6 0.5 N.T. <3 339 739 1.4 0.5 N.T. <3 365 765 1.3 0.4 N.T. <3 35 435 1.2 0.4 N.T. N.T. 176 576 1.2 0.4 N.T. <3 370 770 1.1 0.5 N.T. N.T. 152 552 1.1 0.5 N.T. N.T. 274 674 0.9 0.3 N.T. N.T. 84 484 0.8 0.4 N.T. N.T. 102 502 0.7 0.4 N.T. N.T. 178 578 0.5 0.8 N.T. N.T. 4 404 0.4 0.6 N.T. N.T. Negative control 0.42 0.38 0 <3 S.D. = 0.10 S.D. = 0.06 N.T. indicates not tested

Based on detection of QS-18 2150 and QS-21 1988 components by LCMS/MS (Examples 4-1, 4-2 and 4-3) or UV HPLC quantification of Middle Peak (mainly QS-18 family and desglucosyl-QS-17 family) and Right Peak (mainly QS-21 family) (Example 4-4), Example 4 shows that a number of suitable glucosidases could be identified by screening a set of candidate enzymes (38 from 400, 9.5%). Glucosidases were capable of converting QS-18 family components to QS-21 family components at a range of pHs, concentrations of starting materials and purity of starting materials.

Although certain candidate enzymes did not demonstrate notable conversion under the conditions tested, this may be due to issues with enzyme expression, suitability of conditions (i.e. enzymes may function under other conditions) or a fundamental lack of required enzyme activity.

Example 5—Screening of Additional Glucosidases for Deglycosylation of QS-18 to QS-21 Method Enzyme Selection

Additional candidate glucosidases were selected based on amino acid similarity to an active site model based on positive hits from Example 4.

A final set of 94 additional candidate enzymes was selected. Codon optimized polynucleotide sequences encoding each selected enzyme linked to an N-terminal 6×His tag were prepared. Details of the additional candidate enzyme and polynucleotide sequences are summarised below in Table 8.

TABLE 8 Additional glucosidase candidate sequences AA DNA Seq Seq UniParc Uniprot Organism ID ID UPI000503B26C A0A086YYS8 Bifidobacterium actinocoloniiforme 801 895 UPI000502B461 A0A087CD28 Bifidobacterium psychraerophilum 802 896 UPI00052B8681 A0A0A2K704 Penicillium italicum 803 897 UPI0005ED0AE6 A0A0M2HDB3 Microbacterium trichothecenolyticum 804 898 UPI0008206F38 A0A1C616A3 uncultured Clostridium sp. 805 899 UPI000BC57319 A0A261Y7Q8 Bifiguratus adelaidae 806 900 UPI000B3B73CD A0A378ZIK3 Paenibacillus thiaminolyticus 807 901 UPI000F8FB9BA A0A3S9WE68 Microbacterium lemovicicum 808 902 UPI00019923DC C1A1N4 Rhodococcus erythropolis 809 903 UPI00022C19B5 G4CZU8 Cutibacterium avidum 810 904 UPI0002C5A938 M5BD10 Clavibacter michiganensis 811 905 UPI0003DE3509 W0ZC23 Microbacterium sp. 812 906 UPI000507F38A A0A086YZL4 Bifidobacterium actinocoloniiforme 813 907 UPI000506119A A0A087CR26 Bifidobacterium reuteri 814 908 UPI0005A5CAC2 A0A0A8RX48 Propionibacterium freudenreichii 815 909 UPI0005EC18F9 A0A0M2HRU0 Microbacterium hydrocarbonoxydans 816 910 UPI00094B6D3B A0A1Q8KSV8 Pseudonocardia sp. 817 911 UPI000BC13DF0 A0A292PKV5 Tuber aestivum 818 912 UPI000E5B4EA5 A0A383S7A9 Propionibacterium australiense 819 913 UPI001004B2C9 A0A428T6E2 Fusarium sp. 820 914 UPI0001B69B5C C7ZBV0 Nectria haematococca 821 915 UPI00023EBB15 G8S3E7 Actinoplanes sp. 822 916 UPI0003289BC6 N0CMW2 Streptomyces fulvissimus 823 917 UPI0003E02BF1 W3WXF1 Pestalotiopsis fici 824 918 UPI0005025F2E A0A086ZGP0 Bifidobacterium bohemicum 825 919 UPI0005084E52 A0A087D0Q2 Bifidobacterium saeculare 826 920 UPI00059674D6 A0A0B7JW24 Bionectria ochroleuca 827 921 UPI0006C6D6F9 A0A174AU04 Bifidobacterium pseudocatenulatum 828 922 UPI00094ABAB3 A0A1Q8LPB4 Pseudonocardia sp. 829 923 UPI000CB49A0B A0A2H5Z8Q4 bacterium 830 924 UPI000E38A995 A0A3D8Q771 Coleophoma crateriformis 831 925 UPI001102DFA3 A0A4Z0Y5Y8 Arthrobotrys oligospora 832 926 UPI0001ECDCBD E3RFS2 Pyrenophora teres 833 927 UPI00024F2A26 H6MTQ7 Gordonia polyisoprenivorans 834 928 UPI0000E28E5D Q08S21 Stigmatella aurantiaca 835 929 UPI0003E03A52 W3WZ03 Pestalotiopsis fici 836 930 UPI0003B7B6EE A0A087BEN9 Bifidobacterium magnum 837 931 UPI000503F283 A0A087DFL8 Bifidobacterium stellenboschense 838 932 UPI00059673EE A0A0B7K316 Bionectria ochroleuca 839 933 UPI0006C069F3 A0A174LVE3 Hungatella hathewayi 840 934 UPI00097EB800 A0A1R4J2F9 Mycetocola reblochoni 841 935 UPI0009ABD7B3 A0A2P9IX34 Nonomuraea sp. 842 936 UPI000E391DF6 A0A3D8T9C2 Coleophoma crateriformis 843 937 UPI0000EC83AE A1R2K1 Paenarthrobacter aurescens 844 938 UPI0001F21F08 E4N4F6 Kitasatospora setae 845 939 UPI00027E251E J7L3Z8 Nocardiopsis alba 846 940 UPI0000DBA338 Q0SCI4 Rhodococcus jostii 847 941 UPI0003EC942D W5X324 uncultured microorganism 848 942 UPI0005084B63 A0A087BJ88 Bifidobacterium merycicum 849 943 UPI0005018B5B A0A087DGT3 Bifidobacterium scardovii 850 944 UPI00021EC81E A0A0C4DJL4 Fusarium oxysporum 851 945 UPI0007CE199C A0A177BXH1 Paraphaeosphaeria sporulosa 852 946 UPI0009A50F8D A0A1V4IY77 Clostridium oryzae 853 947 UPI000D22F5EB A0A2T2N4T6 Corynespora cassiicola 854 948 UPI000F7332F8 A0A3N4J9R6 Choiromyces venosus 855 949 UPI0000F550EC A4F982 Saccharopolyspora erythraea 856 950 UPI000204906E F2R0Y2 Streptomyces venezuelae 857 951 UPI00027DFDB0 J7LVB1 Arthrobacter sp. 858 952 UPI00033420CB R7RMQ6 Thermobrachium celere 859 953 UPI0003EA65B9 W7HT93 Drechslerella stenobrocha 860 954 UPI0003B38542 A0A087BMR5 Bifidobacterium minimum 861 955 UPI000409825E A0A087E3K8 Bifidobacterium thermacidophilum 862 956 UPI0005C0229F A0A0D1X9G0 Verruconis gallopava 863 957 UPI0007CECAD5 A0A178AER7 Stagonospora sp. 864 958 UPI0009D58C49 A0A1V6ALF9 Firmicutes bacterium 865 959 UPI000D50EED1 A0A2T6ZNU7 Tuber borchii 866 960 UPI000F728167 A0A3N4KUR3 Morchella conica 867 961 UPI00018152EB B5YC96 Dictyoglomus thermophilum 868 962 UPI000210C01C F5XL24 Microlunatus phosphovorus 869 963 UPI00028BB1B6 K4IPD2 Bifidobacterium asteroides 870 964 UPI00035AE576 S8BQ60 Dactylellina haptotyla 871 965 UPI0003F2D7DD W9JF54 Fusarium oxysporum 872 966 UPI0005067FF9 A0A087BRY2 Bifidobacterium longum 873 967 UPI0004105550 A0A087EK01 Bifidobacterium tsurumiense 874 968 UPI0005BF9DF9 A0A0D1YG50 Exophiala spinifera 875 969 UPI0007CE7DBA A0A178E511 Pyrenochaeta sp. 876 970 UPI0009CDA3F1 A0A1V6FLK2 Firmicutes bacterium 877 971 UPI000D5A9E68 A0A2V1B599 Cadophora sp. 878 972 UPI000F7330CD A0A3N4L4M3 Morchella conica 879 973 UPI000189C68F B8DV42 Bifidobacterium animalis 880 974 UPI00020ED2C9 F5YGD5 Treponema azotonutricium 881 975 UPI0002988429 K7S0E5 Acidipropionibacterium acidipropionici 882 976 UPI0003D93613 V5WNB6 Salinispira pacifica 883 977 UPI000503588E A0A087C760 Bifidobacterium mongoliense 884 978 UPI000499F5D1 A0A087VUW4 Bifidobacterium indicum 885 979 UPI000659257F A0A0G4K5C2 Brachyspira suanatina 886 980 UPI0007F2FD44 A0A194X8Q9 Phialocephala scopiformis 887 981 UPI000A2BB4A5 A0A1Y5P895 uncultured Microbacterium sp. 888 982 UPI000D5BC9BD A0A2V1E673 Periconia macrospinosa 889 983 UPI000F3DC30B A0A3P5WTE2 Arthrobacter ulcerisalmonis 890 984 UPI0001664880 B8H9A2 Pseudarthrobacter chlorophenolicus 891 985 UPI0002120C63 F7PUF3 Haloplasma contractile 892 986 UPI0002B73341 M2ULB2 Cochliobolus heterostrophus 893 987 UPI0003DE58EA W0Z818 Microbacterium sp. 894 988 Experiment 5-1—Screening of Additional Glucosidases for Deglycosylation with Purified QS-18 (0.04 mg/ml) at pH 7.5 and 30 Deg C.

The 94 additional genes, together with positive control (DNA encoding SEQ ID No. 262) and negative control, were transformed, expressed, lysed and reacted in the same manner as described above for Experiment 4-1, except the reaction was incubated at 30 degrees C. for 18 hours.

Samples were analysed by LCMS/MS according to the procedure in Experiment 4-1. The results for all enzymes demonstrating a % conversion of at least 3 are shown in Table 9.

Experiment 5-2—Screening of Additional Glucosidases for Deglycosylation with QS-18 in Crude Bark Extract (80%) at pH 6 and 35 Deg C.

A plate was lysed at pH 6 as described in Experiment 4-2. 40 uL of clarified lysate was transferred to a reaction plate

The pH of CBE was adjusted to pH 6 by dropwise addition of 2M NaOH with stirring. 160 uL of pH 6 CBE was added to each well of the reaction plate. The reaction plate was sealed and incubated at 35 deg C. with shaking for 18 hours.

The reaction plate was quenched by adding 200 uL of MeCN (2% acetic acid (AcOH), 1 mg/mL hexanophenone) to each well of the plates. The quenched reaction plate was re-sealed and incubated at 20 deg C. with shaking for 10 min. The reaction plate was then centrifuged (10 min, 4 degC).

200 uL was transferred from each well of the quenched plate to the corresponding wells of a fresh 96 well plate and sealed. The plate was analysed by UV HPLC with the method of Experiment 4-4.

Experiment 5-3—Screening of Additional Glucosidases for Deglycosylation with QS-18 in Treated Bark Extract (80%) at pH 6 and 35 Deg C.

Experiment 5-1 was repeated, replacing CBE with Treated Bark Extract (TBE) at pH 6. TBE was prepared from CBE by PVPP treatment and concentration, to provide TBE with a QS-21 concentration of approximately 4 g/L. TBE was adjusted to pH 6 by dropwise addition of 2M NaOH with stirring.

TABLE 9 AA DNA Experiment 5-1 Experiment 5-2 Experiment 5-3 Seq ID Seq ID % conv % conv % conv 262 662 98.7 5.5 8.5 850 944 95.3 <0.5 <0.5 879 973 90.1 <0.5 <0.5 868 962 89.9 <0.5 <0.5 826 920 86.9 0.8 1.4 804 898 83.2 <0.5 <0.5 888 982 78.0 <0.5 <0.5 881 975 77.5 <0.5 <0.5 891 985 74.7 <0.5 <0.5 816 910 73.5 <0.5 <0.5 827 921 69.2 <0.5 <0.5 857 951 69.0 <0.5 <0.5 853 947 67.7 <0.5 2.5 842 936 64.0 <0.5 <0.5 814 908 58.4 <0.5 <0.5 886 980 55.5 <0.5 <0.5 885 979 54.8 <0.5 <0.5 838 932 53.4 <0.5 <0.5 829 923 52.3 <0.5 <0.5 808 902 52.0 <0.5 <0.5 828 922 50.7 <0.5 <0.5 870 964 49.7 <0.5 <0.5 873 967 43.9 <0.5 <0.5 844 938 43.6 <0.5 <0.5 882 976 31.4 <0.5 <0.5 874 968 29.4 <0.5 <0.5 825 919 24.5 <0.5 <0.5 824 918 23.5 <0.5 <0.5 823 917 23.1 <0.5 <0.5 810 904 22.7 <0.5 <0.5 894 988 21.3 <0.5 <0.5 849 943 17.8 <0.5 <0.5 803 897 13.1 <0.5 <0.5 890 984 12.6 <0.5 <0.5 841 935 12.3 <0.5 <0.5 832 926 9.8 <0.5 <0.5 830 924 9.3 <0.5 <0.5 845 939 9.2 <0.5 <0.5 871 965 7.8 <0.5 <0.5 837 931 7.6 <0.5 <0.5 883 977 7.0 <0.5 <0.5 809 903 6.3 <0.5 <0.5 875 969 4.7 <0.5 <0.5 862 956 4.2 <0.5 <0.5 864 958 3.9 <0.5 <0.5 848 942 3.8 <0.5 <0.5 805 899 3.8 <0.5 <0.5 854 948 3.7 <0.5 <0.5 840 934 3.5 <0.5 <0.5 817 911 3.5 <0.5 <0.5 819 913 3.2 <0.5 <0.5 876 970 3.1 <0.5 <0.5 Negative control 0.8 <0.5 <0.5

Based on detection of QS-18 2150 and QS-21 1988 components by LCMS/MS (Example 5-1) or UV HPLC quantification of Middle Peak (mainly QS-18 family and desglucosyl-QS-17 family) and Right Peak (mainly QS-21 family) (Examples 5-2 and 5-3), Example 5 shows that a number of suitable glucosidases could be identified by screening a set of candidate enzymes, and also that candidate enzymes demonstrating similarity to previously identified suitable glucosidases were more likely to also be suitable glucosides (51 from 94, 54%). Glucosidases were capable of converting QS-18 family components to QS-21 family components at a range of pHs, concentrations of starting materials and purity of starting materials.

Again, although certain candidate enzymes did not demonstrate notable conversion under the conditions tested, this may be due to issues with enzyme expression, suitability of conditions (i.e. enzymes may function under other conditions) or a fundamental lack of required enzyme activity.

Example 6—Screening of Rhamnosidases for Derhamnosylation of QS-17 to QS-18 Method Enzyme Selection

Conversion of QS-17 family components to QS-18 family components involves hydrolysis of the 1,2 glycosidic bond between the alpha-L-arabinofuranose and alpha-L-rhamnose found at the terminus of the acyl chain portion of the molecules. Glycoside hydrolases from families 78 and 106 exhibit the exo-alpha-1,2 rhamnosidase activity (E.C. 3.2.1.40) necessary to cleave this bond as annotated by the CAZy (Carbohydrate Active enZyme) database (www.cazy.com). All sequences annotated by CAZy from GH families 78 and 106 were obtained, and separate curated hidden Markov model profiles constructed for each which were then used to identify additional familial enzymes by searching the 209 million protein member Uniprot (www.uniprot.org) knowledgebase with the software HMMER (Eddy, 1998). In total, 11,749 sequences were identified: 10,653, and 1096 representatives from GH families 78 and 106, respectively. MMSeqs2 (Hauser, 2016) was then used to cluster each group of enzyme sequences using the default clustering workflow and parameters with a minimum sequence identity and coverage of 30% and 80%, respectively. In cases where the initial clustering yielded clusters with more than 1000 members, a second sub-clustering was performed at a higher 50% or 70% identity to ensure diverse exemplars from these larger clusters were represented more prominently. All clusters were then examined, and exemplars selected from each with preferences for annotation quality, known experimental activity, existing three dimensional structures from the Protein Data Bank (www.wwpdb.org) or known extremophile organisms as annotated by Uniprot. A final set of 94 diverse candidate enzymes was selected. Polynucleotide sequences encoding each selected enzyme linked to a C-terminal 6×His tag and Tev-cleavage site (amino acid sequence for linker His-tag, inserted N-terminally of stop codon, is provided in SEQ ID No. 1178) were prepared using a proprietary genetic-algorithm based codon optimization code.

Details of the candidate enzyme and polynucleotide sequences are summarised below in Table 10.

TABLE 10 Rhamnosidase candidate sequences AA DNA Seq Seq UniParc Uniprot Organism ID ID UPI0001A31108 D0MFR0 Rhodothermus marinus 989 1083 UPI0001D90BFE D7C463 Streptomyces bingchenggensis 990 1084 UPI0001A3AEAC D2QL60 Spirosoma linguale 991 1085 UPI0001CD6D48 D4L2K8 Roseburia intestinalis 992 1086 UPI000442EF22 X5DG83 Draconibacterium orientale 993 1087 UPI00019E052B C7QC24 Catenulispora acidiphila 994 1088 UPI000005ADE1 Q8A916 Bacteroides thetaiotaomicron 995 1089 UPI000172B2E3 B1ZRE4 Opitutus terrae 996 1090 UPI00015FE0BE A9KJP8 Lachnoclostridium phytofermentans 997 1091 UPI00022DA3ED M4NH01 Rhodanobacter denitrificans 998 1092 UPI0001D07633 D5ETD9 Prevotella ruminicola 999 1093 UPI000259E388 I0AZ41 Aspergillus terreus 1000 1094 UPI0001A461B6 C7MA58 Brachybacterium faecium 1001 1095 UPI00006E4A0D A5FCH3 Flavobacterium johnsoniae 1002 1096 UPI000245C507 H2IYR2 Rahnella aquatilis 1003 1097 UPI0003E4E711 W4N6H0 Bifidobacterium moukalabense 1004 1098 UPI000353BD9F S4BBS9 Enterococcus casseliflavus 1005 1099 UPI0001789C0D D3EED1 Geobacillus sp. 1006 1100 UPI000260A2FE 14EYD9 Modestobacter marinus 1007 1101 UPI00019EF0EE C6XYM6 Pedobacter heparinus 1008 1102 UPI00019B5915 C6VZL3 Dyadobacter fermentans 1009 1103 UPI000212C156 F8FQQ3 Paenibacillus mucilaginosus 1010 1104 UPI0003E2544D W4D866 Paenibacillus sp. 1011 1105 UPI000005ADD2 A0A0P0FM19 Bacteroides thetaiotaomicron 1012 1106 UPI00005BA60B A9WDK5 Chloroflexus aurantiacus 1013 1107 UPI00000B098C Q9S3L0 Thermoclostridium stercorarium 1014 1108 UPI0003E5C314 W4N612 Bifidobacterium moukalabense 1015 1109 UPI0003AE032C U2USP4 Olsenella profusa 1016 1110 UPI00019BDB13 D2PMT5 Kribbella flavida 1017 1111 UPI00000C7226 Q9A9K2 Caulobacter vibrioides 1018 1112 UPI000005BA09 Q8A076 Bacteroides thetaiotaomicron 1019 1113 UPI0003745394 U5BUY4 Rhodonellum psychrophilum 1020 1114 UPI0004F6D660 A0A089M3T2 Paenibacillus sp. 1021 1115 UPI0003ED860D W7QMH5 Catenovulum agarivorans 1022 1116 UPI000217D8B1 G0L382 Zobellia galactanivorans 1023 1117 UPI000005B845 Q8A1H5 Bacteroides thetaiotaomicron 1024 1118 UPI0001CD02E9 D6CYE5 Bacteroides xylanisolvens 1025 1119 UPI00018E07C6 B8HAH3 Pseudarthrobacter chlorophenolicus 1026 1120 UPI0001815896 B5YC64 Dictyoglomus thermophilum 1027 1121 UPI00039231C1 T2KPL4 Formosa agariphila 1028 1122 UPI0000DBA6EB Q0S9T4 Rhodococcus jostii 1029 1123 UPI0001D10896 D5GZ45 Lactobacillus crispatus 1030 1124 UPI0001B17DE6 C6XVU2 Pedobacter heparinus 1031 1125 UPI0001A3C989 D2QUA5 Spirosoma linguale 1032 1126 UPI00019EE3EB C6XU05 Pedobacter heparinus 1033 1127 UPI0003432C11 R9ULQ4 Paenibacillus mucilaginosus 1034 1128 UPI0001BC0C05 D5VGD9 Caulobacter segnis 1035 1129 UPI0001969377 E2N9B1 Bacteroides cellulosilyticus 1036 1130 UPI0001B17C60 C6Y153 Pedobacter heparinus 1037 1131 UPI000571C0C2 T2KNB2 Formosa agariphila 1038 1132 UPI00004C6D41 Q5FJ31 Lactobacillus acidophilus 1039 1133 UPI00001AC07D Q7UYD5 Rhodopirellula baltica 1040 1134 UPI0001BF9A6C E3IY10 Frankia inefficax 1041 1135 UPI0001B7FF91 C9Z376 Streptomyces scabiei 1042 1136 UPI00006E5F74 A5FC22 Flavobacterium johnsoniae 1043 1137 UPI00034E666D S2YWB5 Streptomyces sp. 1044 1138 UPI000198DF25 C1F149 Acidobacterium capsulatum 1045 1139 UPI0003ED82D3 W7QYP5 Catenovulum agarivorans 1046 1140 UPI0001A461B7 C7MA59 Brachybacterium faecium 1047 1141 UPI000243A177 A0A0J9X262 Klebsiella oxytoca 1048 1142 UPI0001B24769 C7PA70 Chitinophaga pinensis 1049 1143 UPI0002BCAF6B M3FYL9 Streptomyces bottropensis 1050 1144 UPI0001966B28 D1PKC7 Subdoligranulum variabile 1051 1145 UPI0001F8A51D E8NDD8 Microbacterium testaceum 1052 1146 UPI000053767B Q01V09 Solibacter usitatus 1053 1147 UPI0001A3EFE6 D2B240 Streptosporangium roseum 1054 1148 UPI0006CE1E82 A0A0N1BME3 alpha proteobacterium 1055 1149 UPI000247229E H8KPI7 Solitalea canadensis 1056 1150 UPI0002CB9583 S0GSF0 Parabacteroides goldsteinii 1057 1151 UPI00021B9B33 G0J630 Cyclobacterium marinum 1058 1152 UPI0000533669 Q01TX2 Solibacter usitatus 1059 1153 UPI00032D8F6D R7ZW70 Lunatimonas lonarensis 1060 1154 UPI0000D713F2 Q1M7P3 Rhizobium leguminosarum 1061 1155 UPI0001A3EBEB D2AYU9 Streptosporangium roseum 1062 1156 UPI000156F115 A6LBL4 Parabacteroides distasonis 1063 1157 UPI0003375A10 R9K6L6 Lachnospiraceae bacterium 1064 1158 UPI0001A2F0FA C7P9Y8 Chitinophaga pinensis 1065 1159 UPI0001BC0C15 D5VGC3 Caulobacter segnis 1066 1160 UPI00019EF6E1 C6Y145 Pedobacter heparinus 1067 1161 UPI00019EE1A9 C6Y2X3 Pedobacter heparinus 1068 1162 UPI000C8D4928 A0A2D5SK32 Deltaproteobacteria bacterium 1069 1163 UPI00019BFDCE D1CHL4 Thermobaculum terrenum 1070 1164 UPI000172B62A B1ZY35 Opitutus terrae 1071 1165 UPI00019BFABB D2PXQ4 Kribbella flavida 1072 1166 UPI0001B80091 C9Z391 Streptomyces scabiei 1073 1167 UPI00023EC5D1 G8S540 Actinoplanes sp. 1074 1168 UPI0003C3CD2B V4NSJ1 Asticcacaulis sp. 1075 1169 UPI00019BF65D D2PT74 Kribbella flavida 1076 1170 UPI00000BC760 Q93RE7 Bacillus sp. 1077 1171 UPI00006E5FAB A5FCG3 Flavobacterium johnsoniae 1078 1172 UPI00032EEB9C R7ZS84 Lunatimonas lonarensis 1079 1173 UPI0004B2D794 A0A3E3IGR6 Eisenbergiella massiliensis 1080 1174 UPI0003ED7515 W7QF25 Catenovulum agarivorans 1081 1175 UPI0000184198 Q82PP4 Streptomyces avermitilis 1082 1176

Experiment 6-1—Screening of Rhamnosidases for Derhamnosylation of Saponins in Treated Bark Extract (2%)

Synthetic nucleotide sequences corresponding to SEQ ID 989 to 1082 were subcloned, transformed, expressed and lysed in an identical manner to Experiment 4-1 with the exception that a single cell pellet plate was lysed with 200 ul lysis buffer.

Treated bark extract (TBE) solution was prepared by diluting 1 volume with 9 volumes of 100 mM potassium phosphate pH 7.5. 40 uL clarified lysate was transferred into fresh 96 well PCR plates. 10 uL 10× diluted TBE solution added to each well of lysate to a final concentration of 2% ( 1/50). Plates were incubated at 30 degC with shaking for 18 h. Quenched with 50 uL MeCN+2% AcOH and shaken at room temperature for 10 mins prior to centrifugation (10 min, 4 degC) to remove particulates. Samples were analysed by LCMS/MS using method of Experiment 4-1. The following MS-MS transitions were monitored to observe loss of rhamnose from starting saponins to product derhamnosylated saponins.

Dwell Cone Collision Time MRM Voltage Energy Component Mode (secs) Transitions (V) (v) QS-17 2296 ESI - Auto 1147.52 > 836.40 34 16 QS-18 2150 ESI - Auto 1074.50 > 836.40 34 16 desglucosyl- ESI - Auto 1066.49 > 755.33 34 16 QS-17 2134 QS-21 1988 ESI - Auto 993.46 > 755.5 34 16 QS-17 2310 ESI - Auto 1154.53 > 843.37 34 16 QS-18 2164 ESI - Auto 1081.50 > 843.37 34 16

FIGS. 14 to 19 provide exemplary chromatograms following negative control treatment and rhamnosidase SEQ ID No. 1017 treatment.

Data is expressed as TIC peak area ratio percent (PAR %) for rhamnosylated starting saponin to derhamnosylated product:

${{PAR}\%} = \frac{100 \times {rhamnosylated}{starting}{saponin}}{\begin{matrix} \left( {{{rhamnosylated}{starting}{saponin}} +} \right. \\ \left. {{derhamnosylated}{product}{saponin}} \right) \end{matrix}}$

Activity was measured for the removal of the alpha-O-rhamnosylation at the C2 position of the arabinofuranose moiety of QS-17 2296 component to produce QS-18 2150 component; desglucosyl-QS-17 2134 to produce QS-21 1988 component and QS-17 2310 component to produce QS-18 2164.

No activity was detected for the removal of the C3 saccharide rhamnose as demonstrated by no effect on QS-18 2164 component (QS-18 2164/QS-21 1988 ratio is unchanged). Additionally no endo cleavage of the C28 saccharide rhamnose attached to the C2 position of the fucose was observed.

TABLE 11 AA Seq DNA QS-17 Peak75 Peak57 ratio ID Seq ID (PAR %) (PAR %) (PAR %) PP/QS-21 992 1086 0.000 0.000 0.000 0.20 1003 1097 0.000 0.000 0.000 0.19 1052 1146 0.000 0.000 0.000 0.16 1073 1167 0.007 0.000 0.000 0.18 1017 1111 0.017 0.000 0.004 0.19 1055 1149 0.019 0.000 0.000 0.19 1075 1169 0.043 0.000 0.000 0.18 1001 1095 0.075 0.137 0.000 0.19 1007 1101 0.104 0.043 0.009 0.19 1061 1155 0.245 0.230 0.029 0.19 1079 1173 0.315 0.247 0.000 0.18 1027 1121 0.324 0.094 0.053 0.19 1039 1133 0.492 0.683 0.000 0.18 1041 1135 0.507 0.000 0.000 0.16 989 1083 0.516 0.478 0.305 0.18 1053 1147 1.127 0.944 0.550 0.18 1018 1112 1.512 2.651 0.547 0.17 1066 1160 2.194 1.220 0.896 0.18 1082 1176 3.122 1.614 0.000 0.21 1076 1170 3.766 1.961 1.295 0.19 993 1087 3.836 2.211 1.129 0.18 1077 1171 3.964 2.138 2.532 0.18 1046 1140 4.051 2.446 2.480 0.13 1015 1109 4.132 2.969 3.048 0.15 1063 1157 4.201 2.240 2.473 0.17 1054 1148 4.348 2.160 2.487 0.17 1074 1168 4.443 2.312 1.681 0.18 1067 1161 4.455 2.231 2.852 0.17 1033 1127 4.485 2.521 2.927 0.16 1071 1165 4.587 2.574 2.650 0.17 1059 1153 4.613 2.534 2.956 0.16 1064 1158 4.625 2.476 3.009 0.18 1019 1113 4.626 2.982 2.390 0.15 1047 1141 4.669 2.456 3.332 0.17 1057 1151 4.670 2.498 3.213 0.15 1038 1132 4.725 2.512 2.913 0.18 1032 1126 4.730 2.411 3.044 0.17 1045 1139 4.741 2.442 2.865 0.17 997 1091 4.754 2.115 2.906 0.17 1056 1150 4.774 2.416 2.796 0.17 996 1090 4.778 2.221 3.190 0.16 1069 1163 4.786 2.213 2.596 0.17 1080 1174 4.792 2.434 2.882 0.17 1010 1104 4.796 2.559 2.845 0.18 1035 1129 4.825 2.427 2.987 0.17 1034 1128 4.827 2.389 3.034 0.17 1036 1130 4.834 2.444 2.852 0.16 1008 1102 4.834 2.538 2.658 0.17 1042 1136 4.871 2.599 3.383 0.18 1016 1110 4.872 2.498 3.326 0.18 1023 1117 4.916 2.120 2.889 0.17 1062 1156 4.916 2.410 2.941 0.18 1002 1096 4.922 2.538 2.843 0.16 1029 1123 4.926 2.069 3.334 0.16 1037 1131 4.972 2.424 2.764 0.17 1048 1142 5.007 2.441 2.465 0.17 1000 1094 5.010 2.788 3.119 0.16 1065 1159 5.015 2.458 3.194 0.17 1020 1114 5.058 2.360 3.288 0.17 1012 1106 5.068 2.610 3.249 0.16 1004 1098 5.076 2.553 2.942 0.16 1028 1122 5.081 2.885 1.993 0.15 991 1085 5.084 2.453 3.443 0.17 1011 1105 5.089 2.854 3.124 0.19 1043 1137 5.098 2.791 3.119 0.15 998 1092 5.111 2.402 3.326 0.15 1058 1152 5.121 2.398 3.144 0.17 990 1084 5.144 2.151 0.188 0.21 1024 1118 5.145 2.806 2.924 0.17 1060 1154 5.159 2.382 2.888 0.15 1005 1099 5.166 2.767 2.799 0.17 1070 1164 5.168 2.275 3.311 0.18 994 1088 5.177 2.147 3.300 0.16 1022 1116 5.190 2.368 2.882 0.15 1049 1143 5.211 2.417 2.271 0.18 1031 1125 5.218 2.367 3.096 0.17 1078 1172 5.218 2.153 2.788 0.16 995 1089 5.223 2.545 3.002 0.17 1040 1134 5.244 2.372 3.084 0.18 1050 1144 5.251 2.528 2.305 0.17 1026 1120 5.271 2.501 2.755 0.17 1013 1107 5.272 2.388 2.576 0.17 1051 1145 5.289 2.426 2.918 0.16 1014 1108 5.309 2.741 3.359 0.18 1030 1124 5.315 2.127 3.621 0.17 1006 1100 5.339 2.645 3.237 0.18 1081 1175 5.348 2.473 2.624 0.17 1044 1138 5.370 2.274 3.156 0.16 1009 1103 5.408 2.379 3.762 0.16 1072 1166 5.457 2.462 3.245 0.17 1021 1115 5.476 2.296 3.148 0.16 1025 1119 5.495 2.330 2.933 0.18 1068 1162 5.521 2.370 3.156 0.17 999 1093 5.614 2.571 2.714 0.16 Negative Control 1 4.876 2.719 3.061 0.18 Negative Control 2 5.225 2.158 2.429 0.17

Experiment 6-2 Screening of Rhamnosidases for Derhamnosylation of Saponins in Treated Bark Extract (25%)

A subset of rhamnosidases were expressed and lysed as in the method of experiment 6-1.

Treated bark extract (TBE) solution was adjusted to pH 7.4 by addition of NaOH (2M). 75 uL clarified lysate was transferred into fresh 96 well PCR plates. 25 uL TBE solution (pH 7.4) was added to each well of lysate to a final concentration of 25%. Plates were incubated at degC with shaking for 19.5 h.

Quenched with 100 uL MeCN+2% AcOH and shaken at room temperature for 10 mins prior to centrifugation (10 min, 4 degC) to remove particulates. Samples were analysed by UV HPLC using method of Experiment 4-4. Three key peaks of interest are apparent using this chromatography: Left Peak (retention time approximately 2.30-2.35 min) comprising mainly QS-17 family components; Middle Peak (retention time approximately 2.37-2.42 min) comprising mainly QS-18 family components and desglucosyl-QS-17 family components; and Right Peak (retention time approximately 2.44-2.50 min) comprising mainly QS-21 family components. Peak identity was supported by MS/MS.

Enzyme mediated hydrolysis of the alpha-O-rhamnosylation at the C2 position of the arabinofuranose moiety of QS-17 family components leads to a decrease in Left Peak and an increase in Middle Peak due to formation of QS-18 family components. Enzyme mediated hydrolysis of the alpha-O-rhamnosylation at the C2 position of the arabinofuranose moiety of desglucosyl-QS-17 family components leads to a decrease in Middle Peak and an increase in Right Peak due to formation of QS-21 family components.

The relative percentage of each peak was determined. A decrease in Left Peak and a concomitant increase in Middle Peak and Right Peak is observed for enzymes active under these conditions. Results for the tested subset of rhamnosidases are provided below in Table 12.

TABLE 12 AA Seq DNA Left Middle Right ID Seq ID Peak Peak Peak 1017 1111  6% 63% 31% 1073 1167  6% 62% 31% 1003 1097  7% 63% 31% 1055 1149  7% 62% 31% 992 1086  7% 62% 31% 1007 1101  8% 62% 30% 1052 1146  9% 62% 30% 1075 1169  9% 61% 30% 1061 1155 13% 59% 28% 1001 1095 14% 59% 27% 1079 1173 14% 58% 28% 1041 1135 15% 55% 30% 1039 1133 16% 57% 27% 993 1087 18% 56% 27% 1027 1121 18% 55% 27% 1082 1176 19% 55% 26% 989 1083 19% 54% 26% 1053 1147 20% 54% 26% 1066 1160 20% 53% 27% 1018 1112 22% 52% 26% 1076 1170 22% 52% 26% 990 1084 23% 51% 26% 1077 1171 23% 51% 26% Negative control 24% 50% 26%

Example UV HPLC chromatograms are shown in FIG. 20 for SEQ ID No. 1017 treatment and negative control.

Experiment 6-3 Screening of Rhamnosidases for Derhamnosylation of Saponins in Crude Bark Extract (80%)

Selected variants were expressed and lysed as in the method of experiment 6-1.

Crude bark extract (CBE) solution was adjusted to pH 7.4 by addition of NaOH (2M). 20 uL clarified lysate was transferred into fresh 96 well PCR plates. 80 uL CBE solution (pH 7.4) was added to each well of lysate to a final concentration of 80%. Plates were incubated at 30 degC with shaking for 19.5 h.

Quenched with 100 uL MeCN+2% AcOH and shaken at room temperature for 10 mins prior to centrifugation (10 min, 4 degC) to remove particulates. Samples were analysed by UV HPLC using method of Experiment 4-4.

Three key peaks of interest are apparent using this chromatography: Left Peak (retention time approximately 2.30-2.35 min) comprising mainly QS-17 family components; Middle Peak (retention time approximately 2.37-2.42 min) comprising mainly QS-18 family components and desglucosyl-QS-17 family components; and Right Peak (retention time approximately 2.44-2.50 min) comprising mainly QS-21 family components. Peak identity was supported by MS/MS.

Enzyme mediated hydrolysis of the alpha-O-rhamnosylation at the C2 position of the arabinofuranose moiety of QS-17 family components leads to a decrease in Left Peak and an increase in Middle Peak due to formation of QS-18 family components. Enzyme mediated hydrolysis of the alpha-O-rhamnosylation at the C2 position of the arabinofuranose moiety of desglucosyl-QS-17 family components leads to a decrease in Middle Peak and an increase in Right Peak due to formation of QS-21 family components.

The relative percentage of each peak was determined. A decrease in Left Peak and a concomitant increase in Middle Peak and Right Peak is observed for enzymes active under these conditions. Results for the tested subset of rhamnosidases are provided below in Table 13:

TABLE 13 AA Seq DNA Left Middle Right ID Seq ID Peak Peak Peak 1017 1111 17% 56% 27% 1073 1167 20% 52% 28% 992 1086 21% 49% 30% 1007 1101 24% 49% 27% 1052 1146 25% 49% 27% 1079 1173 25% 48% 27% 993 1087 25% 49% 26% 1041 1135 25% 48% 27% 1055 1149 25% 48% 27% 1039 1133 25% 48% 27% 990 1084 25% 47% 28% 1061 1155 26% 48% 27% 1053 1147 26% 48% 26% 1027 1121 26% 48% 26% 1076 1170 26% 48% 27% 1066 1160 26% 48% 27% 1018 1112 26% 48% 26% 1075 1169 26% 47% 27% 1077 1171 26% 48% 27% 1082 1176 26% 47% 27% 1003 1097 27% 47% 26% 989 1083 29% 43% 27% 1001 1095 31% 45% 24% Negative control 27% 48% 26%

Example UV HPLC chromatograms are shown in FIG. 21 for SEQ ID No. 1017 treatment and negative control.

Based on detection of QS-17 2296, QS-17 2310, QS-18 2150, QS-18 2164, desglucosyl-QS-17 2134 and QS-21 1988 components by LCMS/MS (Example 6-1) or UV HPLC quantification of QS-17, QS-18 and QS-21 peaks (Examples 6-2 and 6-3), Example 6 shows that a number of rhamnosidases could be identified by screening a set of candidate enzymes (29 from 94, 31% achieving a QS-17 PAR % of 4.5 or less in Example 6-1). Rhamnosidases were capable of converting QS-17 family components to QS-18 family components and desglucosyl-QS-17 family components to QS-21 family components at a range of concentrations of starting materials and purity of starting materials.

Again, although certain candidate enzymes did not demonstrate notable conversion under the conditions tested, this may be due to issues with enzyme expression, suitability of conditions (i.e. enzymes may function under other conditions) or a fundamental lack of required enzyme activity.

Example 7—Deglucosylation and Derhamnosylation of Saponins in Crude Bark Extract (50%) Method

E. coli cells expressing glucosidase SEQ ID No. 262 (as His-tagged enzyme, DNA SEQ ID No. 662) and separately rhamnosidase SEQ_ID No. 1017 (as His-tagged form, DNA SEQ ID No. 1111) were grown in a fermenter, isolated, lysed, clarified and the resulting lysate lyophilised to yield powder containing each of the expressed enzymes.

500 uL CBE was mixed with 500 uL volume sodium acetate buffer (50 mM, pH 6) containing 30 g/L lyophilised powder containing the glucosidase, and 3 g/L lyophilised powder containing the rhamnosidase, and incubated at 37 degC for 24 hours.

The reaction was quenched by the addition of an equal volume of MeOH and analysed by LC-MS/MS using the method of Experiment 4-1 monitoring the transitions in the table below

TABLE 14 Component MRM Transitions QS-18 2150  1074.49 > 836.355 QS-18 2164 1081.495 > 843.365 QS-17 2296 1147.515 > 836.355 QS-17 2310 1154.525 > 843.365 QS-21 1988  993.46 > 755.33 QS-21 2002 1000.47 > 762.34 desarabinofuranosyl-QS-18 2018 1008.465 > 836.355 desglucosyl-QS-17 2134 1066.49 > 755.33

Results

FIG. 22 provides exemplary LCMS/MS chromatograms for QS-21 1988 component content at TO (Panel A) and at 24 hrs (Panel B). Results for all components monitored are summarised below:

TABLE 15 Peak area Percent change Component at 24 h at 24 h QS-18 2150 1621 −80% QS-18 2164 95 −94% QS-17 2296 0 −100%  QS-17 2310 0 −100%  QS-21 1988 8215 133% QS-21 2002 1359 266% desarabinofuranosyl-QS-18 2018 6 −79% desglucosyl-QS-17 2134 0 −100% 

Components possessing alpha-O-rhamnosylation at the C2 position of the arabinofuranose moiety are reduced below the detection limit while components possessing a glucose moiety show >78% reduction after treatment for 24 h. The corresponding products of selective rhamnose and glucose hydrolysis show substantial increases.

Example 8—Screening of Glucosidase Variants for Deglycosylation of Saponins in Crude Bark Extract Method

Libraries of genetic variants encoding mutations in the wild type Modestobacter marinus glucosidase (SEQ ID No. 262) were prepared using molecular biology techniques, enzymes were prepared linked to an N-terminally located His-tag. Single monoclonal colonies were grown in 400 ul of expression medium and protein expressed. Cell pellets were lysed in 200 ul of the relevant buffer (Table 16)

Lysate was diluted appropriately in the relevant buffer to allow a lysate loading of the indicated % loading (1% loading corresponds to use of 2 ul original lysate in a 200 ul reaction). In some experiments a rhamnosidase was also present during the screening reaction (and also in controls, negating any impact on results).

Crude bark extract (CBE) obtained by aqueous extraction of Quillaja saponaria and containing at least 2.80 mg/ml QS-21 (by HPLC-UV). The pH of CBE was adjusted to pH 6 by dropwise addition of 2M NaOH with stirring. The relevant concentration of the relevant glucosidase was added. The appropriate relative volume of pH 6 CBE (160 ul (for 80%) or 150 ul (for 75%)) was added to each well of the reaction plate. The reaction plate was sealed and incubated at the relevant temperature with shaking overnight for between 18 and 22 hours.

The reaction plate was quenched by adding 200 uL of MeCN (2% AcOH, 1 mg/mL hexanophenone) to each well of the plates. The quenched reaction plate was re-sealed and incubated at 20 deg C. with shaking for 10 min. The reaction plate was then centrifuged (10 min, 4 degC).

200 uL was transferred from each well of the quenched plate to the corresponding wells of a fresh 96 well plate and sealed. The plate was analysed by UV HPLC with the method below:

UV Method EM2020N435545v2_2

Column details ACQUITY UPLC BEH C18 Column, 130 Å, 1.7 μm, 2.1 mm × 50 mm Column temperature 40° C. Mobile phase A 0.05% v/v TFA in Water HPLC Mobile phase B 0.05% v/v TFA in Acetonitrile HPLC Flow rate 1 mL per minute Time % Mobile % Mobile (mins) Phase A Phase B Gradient profile 0.0 60 40 5.5 min gradient 1.0 60 40 2.1 5 95 2.6 5 95 2.7 60 40 3.5 60 40 Detector type UV detector: DAD or VWD Detector wavelength 214 nm Agilent DAD 1260 4.0 nm (default setting) and 1290 Bandwidth Agilent 1290 and 1260 Off DAD reference wavelength Injection volume Default injection volume used is 1 ul

A negative control (a lysate not expressing test enzymes) and a positive control (expressing the parent comparator—wild type or previous variant as appropriate). Fold improvement over parent (FIOP) for the glucosidase (shorter method) is calculated as follows:

% right peak=100*right peak area/(right peak area+left peak area)

Average % right peak area is calculated for negative controls (per plate) and subtracted from all wells to give the increase in % right peak for each well above average negative control Average increase in % right peak is calculated for positive controls per plate

FIOP=observed increase in % right peak divided by average positive control increase

Results

FIG. 23 provides illustrative chromatograms following treatment of CBE with enzymes and for a negative control.

TABLE 16 Mutations cf Screen Lysate CBE Cumulative Enzyme untagged WT temp pH loading loading Buffer FIOP FIOP (cf WT) Rham WT n/a 35 6  20% 80% 100 mM 1 1 None (SEQ ID Potassium No. 262) phosphate G1 T365N 35 6  20% 80% 100 mM 5 5 None (SEQ ID Potassium No. 1179) phosphate G2 T365N, 20 6  10% 80% 100 mM 10 50 None (SEQ ID R357M, Potassium No. 1180) A473F, phosphate L474C, I475F G3 T365N, 20 6 2.50% 80% 100 mM 2.5 125 Present (SEQ ID R357M, Potassium No. 1181) A473F, L474C, phosphate I475F, F44Y, F442Q G4 T365N, 35 6 0.28% 75% 100 mM 2.4 300 Present (SEQ ID R357M, Sodium No. 1182) A473F, L474C, acetate I475F, F44Y, F442Q, V263L, F5411 G5 F44Y, V263L, 35 6 0.14% 75% 100 mM 3 900 Present (SEQ ID R357M, Sodium No. 1183) T365N, acetate F442Q, L474C, I475F, F5411, A355W, L367C, Q396R TBD—to be determined FIOP—fold improvement over parent (i.e. preceding enzyme), Cumulative FIOP is the product of preceding rounds and is nominally the fold improvement over the original wild-type (WT) starting point (however, since conditions change between rounds and WT isn't used as a control Cumulative FIOP is not a direct measure but an estimate).

The following mutations were associated with enzymes demonstrating improved activity in at least one instance:

-   -   F44Y;     -   V60L;     -   G117A;     -   F170N;     -   V263G or V263L;     -   N351H or N351Q;     -   A355H, A355I, A355L, A355M, A355R, A355T or A355W;     -   A356P;     -   R357A, R357C, R357K, R357M or R357Q;     -   G362C;     -   T365A, T365N or T365S;     -   L367C;     -   V394R;     -   V395Y;     -   Q396E, Q396G, Q396N, Q396P, Q396R, Q396S or Q396Y;     -   F430W;     -   R435F;     -   V438T;     -   V440F;     -   F442M or F442Q;     -   G444T;     -   A473F or A473R;     -   L474C, L474I or L474V;     -   I475F;     -   L492C, L492G, L492H, L492I, L492N, L492Q, L492V, L492W or L492Y;     -   Q493F or Q493H;     -   P494H or P494I;     -   S495I, S495K or S495Q;     -   G496P or G496W;     -   D498A, D498E, D498F, D498I, D498K, D498L, D498N, D498P, D498R,         D498S, D498T or D498V;     -   A502R;     -   M504G or M504R;     -   L507A or L507R;     -   T508M;     -   L529M;     -   F535P;     -   A536D or A536E;     -   A537R;     -   F541A, F541I, F541L, F541M or F541V;     -   L542I;     -   Q543G or Q543L;     -   E547L; and     -   Y585W.

Example 9—Screening of Rhamnosidase Variants for Derhamnosylation of Saponins in Crude Bark Extract Method

Libraries of genetic variants encoding mutations in the wild type Kribbella flavida rhamnosidase (SEQ ID No. 1017) were prepared using molecular biology techniques, enzymes were prepared linked to a C-terminally located His-tag. Single monoclonal colonies were grown in 400 ul of expression medium and protein expressed. Cell pellets were lysed in 200 ul of the relevant buffer (Table 17).

Lysate was diluted appropriately in the relevant buffer to allow a lysate loading of the indicated % loading (1% loading corresponds to use of 2 ul original lysate in a 200 ul reaction).

Crude bark extract (CBE) obtained by aqueous extraction of Quillaja saponaria and containing at least 2.80 mg/ml QS-21 (by HPLC-UV) was adjusted to pH 6 by dropwise addition of 2M NaOH with stirring. The relevant concentration of the relevant glucosidase was added. The appropriate relative volume of pH 6 CBE (160 ul (for 80%) or 150 ul (for 75%)) was added to each well of the reaction plate. The reaction plate was sealed and incubated at the relevant temperature and time.

The reaction plate was quenched by adding 200 uL of MeCN (2% AcOH, 1 mg/mL hexanophenone) to each well of the plates. The quenched reaction plate was re-sealed and incubated at 20 deg C. with shaking for 10 min. The reaction plate was then centrifuged (10 min, 4 degC).

200 uL was transferred from each well of the quenched plate to the corresponding wells of a fresh 96 well plate and sealed. The plate was analysed by UV HPLC with the method described in Example 4.

Three key peaks of interest are apparent using this chromatography: Left Peak (retention time approximately 2.30-2.35 min) comprising mainly QS-17 family components; Middle Peak (retention time approximately 2.37-2.42 min) comprising mainly QS-18 family components and desglucosyl-QS-17 family components; and Right Peak (retention time approximately 2.44-2.50 min) comprising mainly QS-21 family components. Peak identity was supported by MS/MS.

Enzyme activity is calculated as the % conversion of the Left Peak present in the crude bark extract:

${\%{conversion}} = {100 \times \frac{\left( {\%{Right}{Peak}} \right)}{\left( {{\%{Left}{Peak}} + {\%{Right}{Peak}}} \right)}}$ where ${\%{Right}} = \frac{100 \times {Right}{Peak}{area}}{\left( {{{Right}{peak}{area}} + {{Left}{Peak}{area}} + {{Middle}{Peak}{area}}} \right)}$

FIG. 24 provides illustrative chromatograms following enzyme treatment of CBE and for a negative control.

TABLE 17 Conc of Diluted CBE lysate in Lysate Conc CBE solution Temp Time reaction volume reaction volume Enzyme (deg C.) (hours) (% v/v) (uL) (% v/v) (uL) Buffer R1 20 22 1.25 40 80 160 100 mM Potassium (SEQ ID phosphate pH 6 No. 1189) R2 20 20 1.25 40 80 160 100 mM Potassium (SEQ ID phosphate pH 6 No. 1190) R3 35 18 0.4 50 75 150 100 mM Sodium (SEQ ID acetate pH 5.8 No. 1191) R4 35 18 0.125 50 75 150 100 mM Sodium (SEQ ID acetate pH 5.8 No. 1192) R5 35 18 0.0625 50 75 150 100 mM Sodium (SEQ ID acetate pH 5.8 No. 1193) R1 (SEQ ID No. 1189)—K219G R2 (SEQ ID No. 1190)—A143P, L214M, K219G, Q921H; R3 (SEQ ID No. 1191)—A143P, L214M, K219G, G357C, Q921H R4 (SEQ ID No. 1192)—A143P, L214M, G215S, G218N, K219G, G357C, G508S, G634A, Q921H R5 (SEQ ID No. 1193)—A143P, L214M, G215S, G218D, K219G, G357C, G508S, G634A, A690C, Q921H

Results

TABLE 18 % conversion FIOP (% % conversion (Right peak/ area increase Cumulative Left Middle Right (Right peak/ Left Peak) variant/pos FIOP from Enzyme Treatment Peak % Peak % Peak % Left Peak) (−neg control) control) WT R1 R1 27.04 45.89 27.08 50.04 2.74 1.7 1.7 Negative 29.36 44.30 26.35 47.30 0.00 control Positive 27.94 45.30 26.76 48.92 1.62 control (WT) R2 R2 22.76 50.97 26.27 53.57 1.09 2.9 2.9 Negative 23.63 50.27 26.10 52.49 0.00 control Positive 23.31 50.55 26.14 52.86 0.38 control (WT) R3 R3 18.85 51.70 29.45 60.97 13.06 3.3 9.4 Negative 28.91 44.49 26.60 47.91 0.00 control Positive 25.57 46.84 27.59 51.90 3.99 control (R2) R4 R4 25.03 47.96 27.00 51.89 5.06 2.3 22.1 Negative 29.62 44.29 26.09 46.84 0.00 control Positive 27.65 45.79 26.56 49.00 2.16 control (R3) R5 R5 26.59 46.48 26.93 50.32 2.62 1.3 28.7 Negative 28.76 45.00 26.23 47.70 0.00 control Positive 27.09 46.12 26.79 49.72 2.02 control (R4) FIOP—fold improvement over parent (i.e. preceding enzyme), Cumulative FIOP is the product of preceding rounds and is nominally the fold improvement over the original WT starting point (however, since conditions change between rounds and WT isn't used as a control Cumulative FIOP is not a direct measure but an estimate).

The following mutations were associated with enzymes demonstrating improved activity in at least one instance:

-   -   (i) A56C     -   (ii) A143P     -   (iii) Q181H, Q181R or Q181S     -   (iv) L214M     -   (v) G215S     -   (vi) F216M     -   (vii) G218D or G218N     -   (viii) K219G     -   (ix) A238M     -   (x) T252Y     -   (xi) T311W     -   (xii) V326C     -   (xiii) G357C     -   (xiv) S369C, S369I, S369K or S369M     -   (xv) I487M, I487Q or I487V     -   (xvi) K492N     -   (xvii) V499T     -   (xviii) G508S     -   (xix) R543C     -   (xx) L557Y     -   (xxi) G634A     -   (xxii) S635N     -   (xxiii) A690C and     -   (xxiv) Q921H.

Example 10—Deglucosylation and Derhamnosylation of Saponins in Crude Bark Extract (50%) Using Engineered Enzymes Method

Lyophilised powders from clarified cell lysate expressing glucosidases (from Example 8 WT glucosidase and engineered glucosidase polypeptides G1 to G5) and rhamnosidases (from Example 9 WT rhamnosidase and engineered rhamnosidase polypeptides R1 to R5) were dissolved in 200 mM sodium acetate aqueous solution at pH 5.8 to prepare the enzyme solutions at 4 fold the final reaction concentration as shown in Tables 19 and 20.

Each glucosidase solution was combined with an equal volume of 200 mM sodium acetate aqueous solution at pH 5.8 and separately with an equal volume of 200 mM sodium acetate aqueous solution at pH 5.8 containing 2 mg/ml rhamnosidase R5. This is a sufficient loading of rhamnosidase to effect complete hydrolysis of the relevant rhamnose moiety within 4 hours.

Each rhamnosidase solution was combined with an equal volume of 200 mM sodium acetate aqueous solution at pH 5.8 and separately with an equal volume of 200 mM sodium acetate aqueous solution at pH 5.8 containing 2 mg/ml glucosidase G5. This is a sufficient loading of glucosidase to effect complete hydrolysis of the relevant glucose moiety within 4 hours.

CBE was adjusted to pH of 6.0 to 6.2 with 2M sodium hydroxide and an equal volume added to the enzyme solution to prepare the reaction mix (i.e. 50% CBE concentration in reaction mix) and the concentration of glucosidase and/or rhamnosidase is shown in Tables 19 and 20. The reaction mix was heated to 35 degC for the time indicated in Tables 19 and 20, after which the reaction was quenched by addition of an equal volume of MeCN containing 2% acetic acid and shaken at room temperature for 10 mins prior to centrifugation (10 min, 4 degC) to remove particulates. Samples were analysed by UV HPLC using method of Experiment 4-4.

Results

The change in composition of the Left, Middle and Right peaks is shown in in Tables 19 and 20. The composition changes by the action of the enzymes depending on the presence or absence of the partner enzyme. The extent of reaction is proportional to the enzyme concentration and the reaction time under these conditions. The tables show data for enzyme concentrations and the reaction times providing for equivalent extents of reaction. The improvement resulting from the mutations introduced for each variant is equal to the fold change in enzyme concentration×time (i.e. fold improvement=(enzyme concentration×time) for preceding variant÷(enzyme concentration×time) for later variant). A cumulative fold improvement over the original enzyme variant is calculated by the product of the individual fold improvements. The glucosidase G5 shows approximately 800 fold improvement over WT glucosidase. The rhamnosidase R5 shows approximately 30 fold improvement over WT rhamnosidase.

Variants G5 and R5 were found to demonstrate activity across a range of reaction conditions from 25 degC to 40 degC, from pH 5 to 7 (maintaining >80% relative activity for pH 5.4 to 6.2, and >50% for pH 5.2 to 7), and with a range of CBE loadings to at least 150% (achieved by redissolving lyophilised CBE in a smaller volume).

TABLE 19 % Cumulative Rham- % change % Fold Fold Glucosidase nosidase % % % change in change improvement in improvement in conc conc Rham- Time Left Middle Right in Left Middle in Right primary enzyme primary enzyme Glucosidase (mg/ml) (mg/ml) nosidase (h) Peak Peak Peak Peak Peak Peak loading × time loading × time Starting CBE (untreated) 30 44 26 Glucosidase in the absence of Rhamnosidase WT 50 0 — 7 23 24 52 −22% −45% 201% 1 1 G1 6.25 0 — 7 28 18 54  −8% −59% 209% 8.0 8 G2 1.56 0 — 7 26 19 55 −15% −57% 213% 4.0 32 G3 0.78 0 — 7 24 22 53 −19% −50% 206% 2.0 64 G4 0.39 0 — 7 23 23 54 −22% −49% 208% 2.0 128 G5 0.1 0 — 4 27 19 54 −11% −56% 208% 6.5 834 Glucosidase in the presence of Rhamnosidase WT 50 0.5 R5 7 16 16 68 −45% −64% 261% 1 1 G1 6.25 0.5 R5 7 18 16 66 −39% −64% 253% 8.0 8 G2 1.56 0.5 R5 7 22 8 70 −26% −82% 269% 4.0 32 G3 0.78 0.5 R5 7 21 9 70 −31% −79% 270% 2.0 64 G4 0.39 0.5 R5 7 22 9 69 −28% −79% 267% 2.0 128 G5 0.1 0.5 R5 4 20 7 73 −34% −83% 280% 6.5 834

TABLE 20 % Cumulative Rham- % change % Fold Fold Glucosidase nosidase % % % change in change improvement in improvement in conc conc Rham- Time Left Middle Right in Left Middle in Right primary enzyme primary enzyme Glucosidase (mg/ml) (mg/ml) nosidase (h) Peak Peak Peak Peak Peak Peak loading × time loading × time Starting CBE (untreated) 30 44 26 Rhamnosidase in the absence of Glucosidase — 0 5 WT 7 18 52 30 −40%  17% 117% 1 1 — 0 2.5 R1 7 19 51 29 −35%  17% 112% 2.0 2 — 0 1.25 R2 7 18 52 29 −39%  19% 113% 2.0 4 — 0 0.63 R3 7 16 53 30 −45%  21% 116% 2.0 8 — 0 0.31 R4 7 16 54 30 −45%  22% 115% 2.0 16 — 0 0.16 R5 7 17 56 28 −45%  27% 106% 1.9 31 Rhamnosidase in the presence of Glucosidase G5 0.5 5 WT 7 18 3 80 −41% −94% 306% 1 1 G5 0.5 2.5 R1 7 18 2 80 −40% −96% 309% 2.0 2 G5 0.5 1.25 R2 7 18 2 80 −40% −96% 309% 2.0 4 G5 0.5 0.63 R3 7 19 0 81 −38% −99% 311% 2.0 8 G5 0.5 0.31 R4 7 17 3 79 −42% −93% 306% 2.0 16 G5 0.5 0.31 R5 4 20 3 77 −34% −93% 296% 1.7 27

Example 11—Deglucosylation and Derhamnosylation of Saponins in Crude Bark Extract (50%) Using Engineered Enzymes Method

Lyophilised powders from clarified cell lysate expressing engineered glucosidase polypeptide G3 from Example 8 and engineered rhamnosidase polypeptide R2 were dissolved in 200 mM sodium acetate aqueous solution at pH 5.8 to a concentration of 3 g/L (glucosidase) and 2 g/L (rhamnosidase). For a 1 L reaction, sodium acetate buffer 200 mM (700 mL) was charged to a stirred reactor. Under constant agitation, glucosidase enzyme powder (2.1 g) and rhamnosidase enzyme powder (1.4 g) were added and agitated for 30 mins until all the enzyme powder was suspended. The resulting enzyme solution (700 mL) was depth filtered (nom. 3-9 μm) and then sterile filtered (0.2 μm).

CBE (700 mL) containing 4.1 g/L QS-21, with a Preceding Peak ratio of 0.25 and a 2018/QS-21 ratio of 0.054 to 0.057 was depth filtered and then sterile filtered (0.2 μm).

Filtered CBE (500 mL) was charged to a stirred reactor, heated to 37 degC and the pH adjusted to pH of 6.0 to 6.2 with 2M sodium hydroxide. Enzyme solution (500 mL) was then charged to the reactor and the solution stirred at 37 degC for 5 hours.

After 5 hours glacial acetic acid was charged into the reaction mixture gradually under moderate agitation to adjust the pH to ˜pH3.8 (target range pH3.5 to 4.0).

The enzyme treated CBE was then purified analogously to the processes provided in Example 3.

Results

The purified saponin extract was determined to contain at least 98% QS-21 group, at least 93% QS-21 main peak, 0.2% 2018 component, 1% or less of largest peak outside the QS-21 group by UV absorbance at 214 nm and wherein the monoisotope of the most abundant species was 1987.9 m/z.

The increase of QS-21 by mass, based on the QS-21 concentration and the sample volumes, shows a 2.6-3.0× increase in the enzyme treated CBE. The increase of % QS-21 (as % of saponins) showed a 3.0-3.1× increase.

Due to the improved saponin profile of the enzyme treated material a greater recovery yield is obtained while remaining within desired specifications (notably during polystyrene and phenyl resin chromatography where a greater proportion of QS-21 can be recovered). Overall dual enzyme treatment was found to result in approx. 5.2- to 5.3-fold increase in yield compared to a conventional (non-enzyme treated) process.

FIG. 25 provides an example HPLC-UV chromatogram of untreated and enzyme treated CBE.

FIG. 26 (full acquisition) and FIG. 27 (zoom) provide example UPLC-UV chromatograms of purified material obtained from untreated and enzyme treated CBE.

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1. A method for making a product saponin, said method comprising the step of enzymatically converting a starting saponin to the product saponin. 2-3. (canceled)
 4. The method according to claim 1, wherein the starting saponin is a quillaic acid glycoside. 5-6. (canceled)
 7. The method according to claim 1, wherein the starting saponin is a QS-18 family component.
 8. The method according to claim 1, wherein the starting saponin is a desglucosyl-QS-17 family component.
 9. The method according to claim 1, wherein the starting saponin is a QS-17 family component.
 10. The method according to claim 1, wherein the starting saponin is a desarabinofuranosyl-QS-18 family component.
 11. The method according to claim 1, wherein the starting saponin is an acetylated desglucosyl-QS-17 family component. 12-17. (canceled)
 18. The method according to claim 1, wherein a single starting saponin is converted to a single product saponin.
 19. The method according to claim 1, wherein a plurality of starting saponins is converted to a plurality of product saponins.
 20. The method according to claim 1, wherein the starting saponin is obtained by extraction from plant material.
 21. (canceled)
 22. The method according to claim 1, wherein the enzymatic conversion involves the removal of a beta-glucose residue by a glucosidase.
 23. The method according to claim 22, wherein the glucosidase comprises an amino acid sequence according to SEQ ID No. 262, 208, 63, 229, 250, 5, 101, 207, 169, 247, 302, 324, 319, 9, 240, 325, 338, 850, 879, 868, 826, 804, 888, 881, 891, 816, 827, 857, 853, 842, 814, 886, 885, 838, 829, 808, 828, 870, 873, 844, 882, 874, 825, 824, 823, 810, 894, 849, 803, 890, 841, 832, 830, 845, 871, 837, 883 or 809 or functional variants thereof.
 24. The method according to claim 1, wherein the step of enzymatic conversion involves the removal of an alpha-rhamnose residue by a rhamnosidase.
 25. The method according to claim 24, wherein the rhamnosidase comprises an amino acid sequence according to SEQ ID No. 992, 1003, 1052, 1073, 1017, 1055, 1075, 1001, 1007, 1061, 1079, 1027, 1039, 1041, 989, 1053, 1018, 1066, 1082, 1076, 993, 1077, 1046, 1015, 1063, 1054, 1074, 1067 or 1033, or functional variants thereof.
 26. (canceled)
 27. A saponin prepared by the method of claim
 1. 28. (canceled)
 29. An adjuvant composition comprising the saponin according to claim
 27. 30. An immunogenic composition comprising the saponin according to claim 27, and an antigen or a polynucleotide encoding an antigen.
 31. (canceled)
 32. An engineered glucosidase polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID No. 262, or a functional fragment thereof, wherein the engineered glucosidase polypeptide includes at least one residue substitution from: F44Y; V60L; G117A; F170N; V263G or V263L; N351H or N351Q; A355H, A355I, A355L, A355M, A355R, A355T or A355W; A356P; R357A, R357C, R357K, R357M or R357Q; G362C; T365A, T365N or T365S; L367C; V394R; V395Y; Q396E, Q396G, Q396N, Q396P, Q396R, Q396S or Q396Y; F430W; R435F; V438T; V440F; F442M or F442Q; G444T; A473F or A473R; L474C, L474I or L474V; I475F; L492C, L492G, L492H, L492I, L492N, L492Q, L492V, L492W or L492Y; Q493F or Q493H; P494H or P494I; S495I, S495K or S495Q; G496P or G496W; D498A, D498E, D498F, D498I, D498K, D498L, D498N, D498P, D498R, D498S, D498T or D498V; A502R; M504G or M504R; L507A or L507R; T508M; L529M; F535P; A536D or A536E; A537R; F541A, F541I, F541L, F541M or F541V; L542I; Q543G or Q543L; E547L; and Y585W.
 33. (canceled)
 34. An engineered rhamnosidase polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID No. 1017, or a functional fragment thereof, wherein the engineered rhamnosidase polypeptide includes at least one residue substitution from: (i) A56C (ii) A143P (iii) Q181H, Q181R or Q181S (iv) L214M (v) G215S (vi) F216M (vii) G218D or G218N (viii) K219G (ix) A23 8M (x) T252Y (xi) T311W (xii) V326C (xiii) G357C (xiv) S369C, S369I, S369K or S369M (xv) I487M, I487Q or I487V (xvi) K492N (xvii) V499T (xviii) G508S (xix) R543C (xx) L557Y (xxi) G634A (xxii) S635N (xxiii) A690C and (xxiv) Q921H.
 35. (canceled)
 36. A polynucleotide comprising a sequence encoding an engineered glucosidase polypeptide according to claim
 32. 37. A polynucleotide comprising a sequence encoding an engineered rhamnosidase polypeptide according to claim
 34. 